Increased neuronal cell death after intermittent hypercapnic hypoxia in the developing piglet brainstem

Abstract

We examined the immunohistochemical expression of caspase-3 (CASP3), active caspase-3 and TUNEL in the normal piglet brainstem at 13–14 days of age and evaluated the effects of exposure to 2 vs. 4 days of intermittent hypercapnic hypoxia (IHH) on their expression. Eight nuclei from the level of the caudal medulla were studied. In control piglets, CASP3 was present in ∼45% of neurons while active caspase-3 and TUNEL were present in ∼5%, indicating that approximately half the neuronal population of the piglet medulla express caspase-3 in a latent state and that only ∼5% undergo ‘normal’ programmed cell death. After 2 days of IHH, CASP3 increased in the nucleus of the solitary tract (NTS), gracile and cuneate nuclei (P<0.05 for all). Active caspase-3 increased in the dorsal motor nucleus of the vagus (DMNV) (P<0.05) but decreased in the lateral reticular nucleus (LRt) (P<0.05), while TUNEL increased in both the DMNV and LRt (P<0.05 for both). After 4 days of IHH, CASP3 remained elevated in the cuneate nucleus (P<0.01) but decreased in the hypoglossal and DMNV (P<0.05) when compared to controls. Active caspase-3 levels were not changed, whereas TUNEL was increased in the DMNV, LRt, and inferior olivary nucleus (P<0.05 for all). These results show that IHH induces neuronal cell death within certain nuclei in the piglet caudal medulla that are functionally important in cardiorespiratory, sleep and arousal control. This could have important implications for clinical conditions including obstructive apnea and prone sleeping as a risk for SIDS.

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.