Rita Machaalani

Effects of postnatal nicotine exposure on apoptotic markers in the developing piglet brain

Exposure to cigarette smoke is a risk factor for the sudden infant death syndrome (SIDS), but the ability to distinguish between the neuropathological effects of pre- versus postnatal exposure is limited in the clinical setting. To test whether postnatal nicotine exposure could contribute to the increased neuronal expression of apoptotic markers that we have previously observed in SIDS infants, as well as including study of gender influences, we developed a piglet model to mimic passive smoking in the early postnatal period. Piglets were exposed to nicotine (2 mg/kg/day infused via an implanted osmotic minipump) within 48 h of birth until the age of 13-14 days, when the brain was collected for study. Four piglet groups included: control females (n=7), control males (n=7), nicotine females (n=7), and nicotine males (n=7). Apoptotic markers included immunohistochemistry for activated caspase-3, and for DNA fragmentation or terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) in seven nuclei of the brainstem caudal medulla and two subregions of the hippocampus (CA4 and dentate gyrus). Among control females compared with males, there was less active caspase-3 and less TUNEL in the dorsal motor nucleus of vagus (DMNV), and there was less TUNEL in the nucleus of the spinal trigeminal tract (NSTT). Compared with controls, nicotine-exposed male piglets had increased TUNEL staining in the cuneate nucleus (P=0.05), and increased active caspase-3 in the hypoglossal, gracile and dentate gyrus (P<0.05 for each). Nicotine-exposed females showed no change in TUNEL staining in any of the nuclei studied, but increased active caspase-3 in the hypoglossal, DMNV and NSTT (P<0.05 for each). These results show for the first time that postnatal nicotine exposure can lead to an increase in apoptotic markers in the brain. In piglets, these effects showed regional and gender-specific differences, suggesting that passive, postnatal nicotine exposure may be responsible for some neuropathological changes observed in infants dying from SIDS.

NMDA receptors in the developing brain and effects of noxious insults.

This review covers normal expression of the NMDA receptor in the fetus and newborn, and then the response of the NMDA receptors within the central nervous system (CNS) during early development, to noxious stimuli. In the research setting, hypoxia is a commonly studied noxious stimulus that has been studied in a variety of contexts, including isolated hypoxia, or hypoxia combined with ischemia or hypercapnia, and delivered in single or repeated doses (intermittent stimuli). We review differences and commonalities between these experimental paradigms, and the sequelae of a common outcome, which is cell death, possibly through excitotoxic mechanisms. Finally, based on current literature, we will examine potential directions for clinical therapeutic interventions. By highlighting knowledge gaps in this field, we hope to encourage future research focusing on clinically relevant problems and outcomes in this area.

Distribution and quantification of NMDA R1 mRNA and protein in the piglet brainstem and effects of intermittent hypercapnic hypoxia (IHH)

The first aim of this study was to determine the cellular distribution of NR1 in the piglet brainstem. Documentation of NR1 mRNA was by non-radioactive in situ hybridisation (non-RISH) and of NR1 protein by immunohistochemistry. We found that all neurons expressed mRNA but not all had NR1 protein. Application of non-RISH has permitted us, for the first time, to document the cellular localization of NR1 mRNA showing that it was present in the cytoplasm and nucleolus of motor neurons but dispersed throughout the cellular compartments of sensory neurons. NR1 protein on the other hand, was localized to the cytoplasm only. The second aim of this study was to quantify the distribution of NR1 mRNA and protein. Using image analysis software, we used optical density (OD) to quantify the non-RISH signal for mRNA and cellular counting for protein (expressed as % positive). Results show that NR1 expression is greater in motor than sensory nuclei; for mRNA: 0.46+/-0.04 vs. 0.31+/-0.02 OD units (P<0.001), for protein: 75.9+/-3.1 vs. 58.4+/-2.5% positive (P<0.001). The third aim of this study was to determine the effects of intermittent hypercapnic hypoxia (IHH) on NR1 expression. Chronic IHH exposure caused differential changes in mRNA and protein expression. NR1 mRNA expression increased while the number of neurons expressing NR1 protein showed no change. This finding suggests that NMDA pathways are activated by exposure to IHH.

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta.

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women.

Brain-derived neurotrophic factor mRNA and protein in the piglet brainstem and effects of Intermittent Hypercapnic Hypoxia.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin essential for the development of normal respiratory rhythm and ventilatory control. Chronic exposure to Intermittent Hypercapnic Hypoxia (IHH) has been shown to alter ventilatory responses of piglets. This study investigated changes in BDNF distribution and expression in seven nuclei of the caudal medulla, from piglets exposed to IHH for 1, 2, 3, or 4 days before death, using non-radioactive in situ hybridisation (for mRNA) and immunohistochemistry (for protein). Compared to controls, BDNF mRNA was markedly increased across the entire medulla of the brainstem, after all durations of IHH (1-4 days). In contrast, BDNF protein expression increased after 1 day of exposure to IHH (p=0.003), but, thereafter, was not different to controls. Amongst individual nuclei, neurons of the dorsal motor nucleus of the vagus (DMNV) showed increased BDNF mRNA (p<0.01), but decreased protein expression (p=0.05) after all durations of IHH. In the ION, both mRNA and protein for BDNF were significantly increased after 1 day IHH (p<0.01 and p=0.001, respectively), but these increases were not sustained. This study is the first to investigate changes in BDNF expression in response to environmental challenges during postnatal development in the brainstem. Implications of the wide distribution of BDNF in the piglet caudal medulla and increased expression after IHH exposure are discussed, with particular reference to roles for BDNF-dependent neurons at this stage of development.

Changes in orexin (hypocretin) neuronal expression with normal aging in the human hypothalamus

Animal studies have shown that decreased orexin expression changes sleep regulation with normal aging. This study examined orexin A and B expression in the tuberal hypothalamus in infants (0-1 year; n = 8), children (4-10 years; n = 7), young adults (22-32 years; n = 4), and older (48-60 years; n = 7) adults. Neuronal expression was defined by the percentage positive orexin immunoreactive (Ox-ir) neurons in the whole tuberal hypothalamus, and in the dorsal medial (DMH), perifornical, and lateral hypothalamus. In addition, the number of Ox-ir neurons/mm(2), regional distribution, and co-localization were examined. Within the whole tuberal hypothalamic section, there was a 23% decrease in the percentage of Ox-ir neurons between infants and older adults (p < 0.001), and a 10% decrease in older compared with younger adults (p = 0.023). These changes were confined to the DMH and/or perifornical hypothalamus. There was a 9%-24% decrease in Ox neurons/mm(2) in adults compared with infants and/or children (p ≤ 0.001). These results demonstrate a decrease in Ox expression with normal human maturation and aging. This may contribute to changes in sleep regulation during development and with aging.

Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits α7 and β2 in the sudden infant death syndrome (SIDS) brainstem

It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared α7 and β2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n=46) and non-SIDS infants (n=14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased α7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased β2 in the cNTS and increased β2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased α7 in the vestibular nucleus and increased β2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for α7 in the gracile and cuneate, and β2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on α7, but prone sleep was associated with decreased β2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of α7 and β2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure (β2), gender (α7 and β2) and sleep position (β2) evident.

Immunolocalization of pro- and mature-brain derived neurotrophic factor (BDNF) and receptor TrkB in the human brainstem and hippocampus

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are essential in promoting normal development of the central nervous system. Specific functions that are affected in knockout models include respiratory control, coordination of movement and balance, and feeding activities. The expression of these markers has not yet been studied in the human infant brain. This study provides a detailed account of the distribution and localization of both pro- and mature-recombinant human (rh) forms of BDNF, and of TrkB in the human infant brainstem and hippocampus, and qualitatively compares this expression to that seen in the human adult. Using commercially available antibodies, we applied immunohistochemistry on formalin fixed and paraffin embedded human brain tissue [n=8 for infant, n=6 for adult], and qualitatively analyzed the expression of proBDNF, rhBDNF and TrkB. Amongst the brainstem regions studied, the greatest expression of the markers was in the mesencephalic trigeminal of the pons, and in the medulla, the inferior olive and arcuate nucleus. The lowest expression was in the substantia nigra of the midbrain and pontine locus coeruleus. Compared to adults, all the studied markers had a higher expression in the infant brainstem nuclei of the hypoglossal, vestibular, dorsal motor nucleus of the vagus, prepositus, cuneate, and dorsal raphe. In the hippocampus, only TrkB showed a higher expression in infants compared to adults. We conclude that BDNF and TrkB play important roles in controlling respiration, movement, balance and feeding in the brainstem and that the TrkB receptor is the most age-sensitive component of this system, especially in the hippocampus.

Active caspase-3 in the sudden infant death syndrome (SIDS) brainstem

In a retrospective postmortem study, we examined the neuronal expression of active caspase-3, a specific apoptotic marker, in the brainstem of 67 infants dying from sudden infant death syndrome (SIDS), and 25 age-matched control infants (non-SIDS). Neuronal immunostaining for active caspase-3 was semi-quantitatively scored in nuclei from five brainstem levels: rostral, mid and caudal pons, and rostral and caudal medulla. Regardless of the cause of death (SIDS vs. non-SIDS), age-related differences in active caspase-3 expression were identified, predominantly in the medulla. No gender-related differences were identified. Comparing SIDS to non-SIDS cases, increased active caspase-3 expression was restricted to four nuclei in the caudal pons (abducens, facial, superior olivary, and pontine nuclei) and two nuclei in the rostral medulla (hypoglossal and dorsal motor nucleus of the vagus). We conclude that neuronal apoptosis is increased in the brainstem of SIDS compared to non-SIDS infants.

Neurochemical abnormalities in the brainstem of the Sudden Infant Death Syndrome (SIDS).

The brainstem has been a focus in Sudden Infant Death Syndrome (SIDS) research for 30 years. Physiological and animal model data show that cardiorespiratory, sleep, and arousal mechanisms are abnormal after exposure to SIDS risk factors or in infants who subsequently die from SIDS. As the brainstem houses the regulatory centres for these functions, it is the most likely site to find abnormalities. True to this hypothesis, data derived over the last 30 years shows that the brainstem of infants who died from SIDS exhibits abnormalities in a number of major neurotransmitter and receptor systems including: catecholamines, neuropeptides, acetylcholinergic, indole amines (predominantly serotonin and its receptors), amino acids (predominantly glutamate), brain derived neurotrophic growth factor (BDNF), and some cytokines. A pattern is emerging of particular brainstem nuclei being consistently affected including the dorsal motor nucleus of the vagus (DMNV), nucleus of the solitary tract (NTS), arcuate nucleus (AN) and raphe. We discuss the implications of these findings and directions that this may lead in future research.