Research reportIncreased neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet brainstem
Introduction
In animal models, stimuli involving hypoxia [2], [3], ischemia [10], [20] and hypoxic ischemia [1], [29] have been shown to precipitate neuronal cell death. Whether this cell death is apoptotic as opposed to necrotic has been the subject of much recent debate [12] mostly because these studies relied on histochemical staining rather than electron microscopic analysis by which apoptosis was originally defined [14]. One method commonly used to identify apoptosis is the terminal deoxynucleotidyltransferase-mediated dUTP–biotin nick end labelling (TUNEL) method. TUNEL staining identifies DNA fragmentation, a feature of a cell in the late stages of apoptosis, but its specificity for apoptosis is often questioned, leading to the recommendation that results be interpreted in conjunction with at least one other apoptotic marker [16].
Activation of the caspases, cysteine proteases, is an essential component of the process of apoptosis [25]. In the brain, caspase-3 is especially important, where it plays a key role in the initiation of the apoptotic pathway [6] and is thought to be responsible for many cytological changes that characterise neuronal apoptosis [4]. Caspase-3 exists as an inactive proenzyme of molecular weight 32 kDa. Noxious stimuli precipitate the cleavage of this proenzyme into two active subunits of molecular weight 12 and 17 kDa, which then associate to form the active caspase-3 enzyme. This active enzyme then cleaves a variety of enzymes and substrates including other caspases initiating the caspase cascade whereafter the cell is committed to die [17]. Thus, caspase-3 is considered an early marker of the apoptotic pathway.
In this study, we used intermittent hypercapnia hypoxia (IHH) as our noxious stimulus. This stimulus was designed to mimic the type of exposure experienced in clinical situations such as obstructive apnea or sleeping prone and face down, a known risk factor for the sudden infant death syndrome (SIDS) [13]. This study follows on from the original observation that apoptotic markers were increased in the brainstem of SIDS infants [26].
A piglet model of IHH has been established in our laboratory, where we have recently demonstrated subsequent functional changes (depression) of the ventilatory responses [27], changes in the expression for the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor [18], and in TUNEL expression amongst NR1 neurons [19] within certain nuclei of the brainstem medulla. The piglet serves as a clinically relevant animal model in developmental studies of cardiorespiratory control mechanisms [7]. Piglets aged 13–14 days were studied because at this age, the structure and functional maturation of the pulmonary vasculature [8], the respiratory pattern behaviour [24] and brain development [5] reflect that of 2–4 month-old infants, a period of highest incidence of SIDS [11].
We hypothesize that a stimulus of IHH during early development of the piglet increases neuronal cell death and that this increase is affected by the cumulative duration of IHH exposure. To test this hypothesis, immunohistochemistry for caspase-3 (CASP3), active caspase-3 and the TUNEL method were employed. Eight nuclei at the level of the caudal medulla were selected for study on the basis of their functional relevance to cardiorespiratory control, sleep and arousal.
Section snippets
Piglet model of IHH
Mixed-breed miniature piglets aged 9–12 days (n=17) were assigned to either control or treatment groups. The assigned study protocol was always undertaken in the period immediately prior to death. The treatment in this study was intermittent hypercapnic hypoxia (IHH) (8% O2, 7% CO2, balance N2) over 48 min, 6 min of hypercapnic–hypoxia (HH) alternating with 6 min of air (total 24 min of HH). Thus, on each study day, a total of 24 min HH was delivered in an intermittent pattern for either 2 or 4
Piglet characteristics
Piglet characteristics are shown in Table 1. All brains were well perfused and collected from piglets of the same age. There was no difference amongst groups for the average daily weight gain or the body weight at death. Brain weights were significantly reduced in the 2dIHH treated group when compared with the control and 4dIHH groups. Brain to body weight ratios were also significantly less in the 2dIHH group compared to the controls and 4dIHH group.
Distribution and pattern of staining
Positive staining was present in neurons and
Discussion
This study examined the expression of caspase-3 (CASP3), active caspase-3 and TUNEL in eight nuclei of the piglet caudal medulla and assessed whether the expressions were changed after 2 and 4 days of IHH exposure. Our results show that IHH induced an increase in the expression of these apoptotic markers, but the two durations of exposure caused different patterns of change. We interpret the pattern of changes to reflect different stages of the cell death cycle, with progression from active
Conclusion
The important finding of this study is that a stimulus of IHH induces an increase in the number of neurons undergoing cell death in the developing piglet brainstem. Although the precise form of cell death could not be determined, two markers for apoptosis, active caspase-3 and TUNEL, were affected, with different degrees depending on the duration of the stimulus. After 2 days, both markers were affected whereas after 4 days, only TUNEL was affected, suggesting that changes in active caspase-3,
Acknowledgements
Research was funded by: NH&MRC #980504, The Ramaciotti Foundation and Financial Markets Trust for Children. Rita Machaalani is a scholarship recipient from Community health and Tuberculosis Association in Australia (CHATA). Dr Waters is supported by NH&MRC Fellowship #206507. The authors would like to thank Kellie Tinworth for the live animal studies, Jane Radford for technical assistance in immunohistochemistry and Dennis Dwarte (Electron Microscopy Unit) for technical assistance in image
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