Elsevier

Neuroscience

Volume 142, Issue 1, 29 September 2006, Pages 107-117
Neuroscience

Cellular neuroscience
Postnatal nicotine and/or intermittent hypercapnic hypoxia effects on apoptotic markers in the developing piglet brainstem medulla

https://doi.org/10.1016/j.neuroscience.2006.06.015Get rights and content

Abstract

The most important risk factors currently identified for the sudden infant death syndrome (SIDS) are prone sleeping and cigarette smoke exposure. In this study, we investigated the neuropathological sequelae of these risk factors by exposing piglets to intermittent hypercapnic-hypoxia (IHH) and/or nicotine (nic) in the early postnatal period. Our hypothesis was that either nic or IHH exposure could increase neuronal cell death, and that combined exposure (nic+IHH) would be additive. Four exposure patterns were studied: controls (n=14), IHH (n=10), nic (n=14), and nic+IHH (n=14). All groups had equal gender ratios. Nic exposure via an implanted osmotic minipump commenced within 48 h of birth and continued until age 13–14 days when animals were killed and brains collected. A total of 48 min of hypercapnic-hypoxia was delivered on the day immediately prior to killing in a pattern comprising 6 min of HH (8% O2, 7% CO2, balance N2) alternating with 6 min of air. Immunohistochemistry was performed to identify neurons positive for active caspase-3 and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL) in seven nuclei of the caudal medulla. Staining quantification showed that: 1. IHH induced neuronal death (increased both TUNEL and casapse-3) in more brainstem nuclei than nicotine. 2. Females were more severely affected by IHH than males. 3. Where IHH and nicotine were combined, TUNEL expression was ∼5% less than IHH alone, but changes in caspase-3 were variable. We conclude that acute exposure to IHH in the postnatal period is more neurotoxic than exposure to nicotine alone. Combined exposure to IHH and nicotine produced variable responses with some results suggesting that nicotine can be neuroprotective. These results indicate that environmental insults attributable to prone sleeping can produce neurotoxic sequelae in SIDS, with some regional specificity in the response. However, no consistent relationship is evident when combining the two insults.

Section snippets

Animal exposures and surgery

Mixed-breed miniature piglets were used in this study. All piglets were received from a commercial piggery within 2 days of birth. Piglets were then randomly assigned to one of the following exposure groups: 1) saline control, 2) nicotine, 3) nicotine and IHH (nic+IHH), or 4) IHH. Piglets in groups 1–3 underwent surgery for implantation of minipumps. Piglets in group 4 had no surgery at all but were exposed to 1 day’s worth of IHH, which was similarly given to piglets in group 3.

Implant of minipumps for nicotine exposure

Osmotic

Piglet characteristics and cotinine concentrations (Table 1)

A total of 52 piglets were studied. Each group had equal numbers of males and females. Across groups and between genders there was no statistically significant difference for any physical parameters including age, body weight at surgery and at death, average daily weight gain, or brain weight (Table 1).

In the group exposed to nicotine only, mean concentrations of cotinine measured at the time of death were 20.3±2.5 ng/ml in serum (males: 19.8±5.0, females: 20.8±1.1), and 92.7±21.8 ng/ml in

Discussion

In this study we found that during early postnatal development: 1. IHH exposure is a greater neurological insult than nicotine alone, 2. when the insults of IHH and nicotine were combined, TUNEL staining increased but to levels that were less than occurred with IHH alone, suggesting that nicotine may be neuroprotective against IHH, and 3. the most striking gender differences were evident for caspase-3 activation after IHH exposure where females were more affected than males, but the apparently

Conclusion

We have shown for the first time that an acute 1 day IHH exposure is more neurotoxic than continuous nicotine infusion over 2 weeks. Compared with our previous data, the peak of apoptotic expression after 1 day of IHH is greater than that seen after 2 or 4 days. Moreover, IHH-induced cell death seems to follow different paths depending on gender, with caspase-3 activation being more prominent in females and apparently activated earlier than in males. Nicotine in the presence of IHH either

Acknowledgments

Research was funded by: NH&MRC #302006. Prof. Waters is supported by an NH&MRC Practitioner Fellowship (#206507). We thank Kellie Tinworth for the care and handling of the piglets during the study period. We also acknowledge the facilities as well as scientific and technical assistance from staff in the NANO Major National Research Facility at the Electron Microscope Unit, the University of Sydney.

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