Suppressed fever and hypersensitivity responses in chicks prenatally exposed to opiates

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Abstract

We have established procedures to reliably induce opiate dependence in the chick embryo via in ovo injection, early in embryonic development, of the long-acting and potent opiate N-desmethyl-l-α-noracetylmethadol (NLAAM). Prior studies found that there is continual exposure to NLAAM throughout embryogenesis and shortly after hatching there are signs of spontaneous withdrawal. In the present study, we used three doses of NLAAM (2.5, 5, and 10 mg/kg egg weight) to determine if prenatal opiate exposure followed by postnatal withdrawal interfered with appropriate neural–endocrine–immune interactions in the young chick. To ensure that effects were not a consequence of inappropriately large doses, we first examined acute and chronic toxicity and additional characteristics of postnatal opiate withdrawal. We then measured the corticosterone and fever responses to LPS stimulation during the withdrawal period. After the conclusion of opiate withdrawal, we assessed the hypersensitivity response to phytohemagglutinin (PHA). The fever response to LPS and the hypersensitivity response to PHA were suppressed by prenatal opiate exposure and postnatal withdrawal. The corticosterone response to LPS was not affected, but there were exaggerated corticosterone responses to saline injection in chicks exposed in ovo to NLAAM. It was unlikely that the effects of prenatal NLAAM were the result of toxicity, as little chronic toxicity was seen with the lower two doses of NLAAM, doses that yielded significant suppressions of neural–endocrine–immune responses. However, effects found in the chicks treated with 10 mg NLAAM/kg may have been partly related to the greater toxicity and/or protracted postnatal withdrawal in this group.

Introduction

Xenobiotic use during pregnancy is associated with medical complications for the mother and fetus. For the fetus, exposure to exogenous compounds that mimic endogenous neurotransmitters (or modulators) can impact not only the developing brain, but also other systems that interact with the nervous system, such as the endocrine and immune systems. Opiates, in particular, are of concern because of their potent immunomodulatory properties and their effects on the hypothalamic–pituitary–adrenal (HPA) axis. It is well established that acute and chronic opiate administration has immunomodulatory effects, particularly with respect to innate immunity and T-cell function (Carr et al., 1996; Eisenstein and Hilburger, 1998; Roy and Loh, 1996). Interestingly, withdrawal from opiates is also characterized by changes in immune function that may be dissociable from direct effects of opiate exposure (Bhargava et al., 1989; Rahim et al., 2002; West et al., 1999). Central processes likely play a role in some of the effects of opiate exposure and withdrawal (e.g., Nelson and Lysle, 2001; Nelson et al., 2000), but interactions between the HPA axis, sympathetic nervous system, and immune system have also been implicated (e.g., Houghtling and Bayer, 2002; West et al., 1999).

While there has been considerable study of the effects of opiate exposure, and to a lesser extent opiate withdrawal, in mature subjects, much less is known about the long-term immunomodulatory consequences of opiate exposure and withdrawal during development.

Shavit et al. (1998) found that morphine administered to pregnant rats blunted natural killer cell activity and an induced fever response, but not the behavioral response to lipolysaccharide (LPS) when the offspring were mature. However, there has been no assessment of immune responding in the neonate following opiate exposure, likely because of the difficulty in assessing these measures in young altricial animals. Neonates are inherently vulnerable to infectious agents because of the immaturity of their immune systems. If opiate exposure or withdrawal compromise the ability of the immune system to respond to infectious challenge, then significant health-related consequences may ensue. This is of particular relevance following prenatal opiate exposure, when infants often have prolonged hospitalization while undergoing a neonatal narcotic abstinence syndrome (e.g., Archie, 1998; Kaltenbach et al., 1998). Thus, in the present study we assessed two measures of immune system reactivity—the fever response to LPS and the cutaneous basophil hypersensitivity response to the T-cell antigen phytohemagglutinin (PHA). The former assessment relies on intact neural–immune functioning. LPS is a component of the cell wall of Gram-negative bacteria. It elicits a profound immune response, with a strong macrophage-mediated inflammatory component (Derijk et al., 1993). As a consequence of the inflammatory cascade, body temperature increases (fever), corticosterone and its antecedent releasing hormones are released, and organism behavior changes (sickness behavior) (e.g., Kent et al., 1992). The cutaneous basophil hypersensitivity response is an example of a delayed-type hypersensitivity reaction, characterized by an inflammatory response to the antigen exposure. There is strong evidence that this response to PHA is mediated by T-cells, since manipulations that reduce T-cell number (Goto et al., 1978) or activity (Corrier and DeLoach, 1990) suppress this hypersensitivity reaction.

The effects of opiate exposure and/or withdrawal on neuroendocrine function may also influence the neonatal immune response. Heightened activity of the HPA axis has been well documented for both opiate tolerance and dependence in mature rodents (see comprehensive review by Pechnick, 1993), with both increasing circulating corticosterone via altering hypothalamic function (e.g., Georges and Way, 1979; Holaday et al., 1979; Milanes et al., 1998). Enhanced emotional reactivity and hyperresponsiveness to environmental change are markers used to identify both human and animal infants undergoing opiate withdrawal. These altered responses suggest an underlying disruption of normal HPA axis function during neonatal withdrawal. Because of the potential long-term effects of altered HPA axis reactivity during the neonatal period on behavioral, neuroendocrine, and immune function (e.g., Denenberg and Zarrow, 1971; Kalinichev et al., 2002; Loizzo et al., 2002; Meaney et al., 1996), it is important to establish whether basal HPA axis function, as well as a response to mild or more severe stressors, are altered during neonatal opiate withdrawal. Thus, in addition to immune measures, basal and stress-induced serum corticosterone was examined in the present study using a chick embryo/chick hatchling model. The domestic chick has features that make it useful for developmental studies of abused drugs. In ovo drug administration is easily accomplished and it eliminates prenatal maternal undernutrition and handling stress. In addition, since the chick is precocial, early postnatal manipulations can be conducted without maternal or littermate influence, a potential source of confounding “early experience” factors in studies of this type. The chick responds to immune system stimulation by LPS in a similar fashion as mammals, with induction of sickness behavior and cognitive deficits, as well as increased body temperature and circulating corticosterone (e.g., Johnson et al., 1993a, Johnson et al., 1993b; Schrott and Sparber, 2001; Schrott et al., 1999; Sell et al., 2001).

We have established procedures to reliably induce opiate dependence in the chick embryo using the long-acting and potent opiate N-desmethyl-l-α-noracetylmethadol (NLAAM). NLAAM is an active metabolite of the opiate l-α-acetylmethadol (LAAM), a synthetic opiate used in substitution therapy for treatment of heroin addiction (Trueblood et al., 1978). In the adult, mixed function oxidative enzymes N-demethylate LAAM to more active metabolites, including NLAAM (Young et al., 1979). However, in the very young chick embryo the metabolic enzymes are not active (Lorr and Bloom, 1987; Mercurio et al., 1983) and to maintain more mature levels of activity throughout embryonic development, we directly administer the most active metabolite, NLAAM. NLAAM is administered in ovo by injection of the drug underneath the shell of the egg into the allantoic fluid on embryonic day (E) 4. The drug is distributed to the embryo via transport through the extra-embryonic vasculature. Studies using [3H]NLAAM demonstrated that this injection procedure results in effective delivery of the drug to the embryo within 24 h (Kuwahara and Sparber, 1981a). Prior studies have established that there is continual exposure to NLAAM throughout embryogenesis with peak concentrations of NLAAM found in the brain one day after hatching. This peak occurs because just prior to hatching there is resorption of the remainder of the yolk sac, a storage site for the drug (Kuwahara and Sparber, 1981a). Shortly after hatching, there are signs of a spontaneous withdrawal syndrome in the hatchling (Kuwahara and Sparber, 1982). In addition, to further establish that chick embryos have become opiate-dependent following in ovo NLAAM administration, we examined antagonist-induced precipitated withdrawal during embryogenesis. We have previously reported that during naloxone-precipitated withdrawal there is increased embryonic motility (Kuwahara and Sparber, 1981b), activation of the HPA axis, and increased blood vessel diameter (Schrott et al., 2002).

In the present set of studies, we used three doses of NLAAM administered on E4. To ensure that effects were not a consequence of an inappropriately large doses, we first examined issues of acute and chronic toxicity associated with prenatal NLAAM exposure, as well as other characteristics of the subsequent postnatal opiate withdrawal. We then assessed HPA axis responsivity and the fever response to LPS during the withdrawal period, and then, after the conclusion of opiate withdrawal, we assessed the hypersensitivity response to PHA.

Section snippets

Subjects

This experiment was carried out in multiple phases in order to generate sufficient embryos and hatchlings for the various dependent measures. Study phase was included as a fixed variable in the statistical analyses. Table 1 depicts the overall design for the study, including the age for each manipulation and the dependent measures examined. Eggs containing viable E4 White Leghorn embryos were obtained from the Poultry Nutrition Research Center at the University of Minnesota (St. Paul, MN). The

Acute and chronic NLAAM toxicity

Acute toxicity was assessed on E8 using the non-invasive candling procedure. There was an overall effect of Prenatal Treatment (χ2(3)=13.05, p<.005). As can be seen in Table 2, all three doses of NLAAM reduced the number of viable embryos by approximately 15% compared to the vehicle-treated controls (χ2(1)=10.98, 6.56, and 9.43, p<.0009, .02, and .003, respectively). There were no differences amongst the NLAAM doses.

To assess chronic toxicity we assessed hatching parameters. There was an

Discussion

In the present study we demonstrated that fever responding to LPS and the hypersensitivity response to PHA were suppressed by prenatal opiate exposure in combination with postnatal withdrawal. The corticosterone response to LPS was not affected, but there were altered corticosterone responses to saline injection in NLAAM-treated offspring that may have interfered with LPS responses, particularly in the chicks exposed to the higher NLAAM doses in ovo. While it is not possible to completely rule

Acknowledgements

This work was supported, in part, by USPHS Grants K01 DA00362 (L.M.S.) and R37 DA04979 (S.B.S.). We thank Ms. Erin Larson and Ms. Ann Choi for their technical assistance.

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