Central and peripheral chemoreceptors in sudden infant death syndrome

Abstract

The pathogenesis of sudden infant death syndrome (SIDS) has been ascribed to an underlying biological vulnerability to stressors during a critical period of development. This paper reviews the main data in the literature supporting the role of central (e.g. retrotrapezoid nucleus, serotoninergic raphe nuclei, locus coeruleus, orexinergic neurons, ventral medullary surface, solitary tract nucleus) and peripheral (e.g. carotid body) chemoreceptors in the pathogenesis of SIDS. Clinical and experimental studies indicate that central and peripheral chemoreceptors undergo critical development during the initial postnatal period, consistent with the age range of SIDS (<1 year). Most of the risk factors for SIDS (gender, genetic factors, prematurity, hypoxic/hyperoxic stimuli, inflammation, perinatal exposure to cigarette smoke and/or substance abuse) may structurally and functionally affect the developmental plasticity of central and peripheral chemoreceptors, strongly suggesting the involvement of these structures in the pathogenesis of SIDS. Morphometric and neurochemical changes have been found in the carotid body and brainstem respiratory chemoreceptors of SIDS victims, together with functional signs of chemoreception impairment in some clinical studies. However, the methodological problems of SIDS research will have to be addressed in the future, requiring large and highly standardized case series. Up‐to‐date autopsy protocols should be produced, involving substantial, and exhaustive sampling of all potentially involved structures (including peripheral arterial chemoreceptors). Morphometric approaches should include unbiased stereological methods with three‐dimensional probes. Prospective clinical studies addressing functional tests and risk factors (including genetic traits) would probably be the gold standard, allowing markers of intrinsic or acquired vulnerability to be properly identified.

Introduction

According to the so‐called ‘triple‐risk model’, the pathogenesis of sudden infant death syndrome (SIDS) is considered to be the result of underlying biological vulnerability to stressors during a period of critical development (Filiano & Kinney, 1994). Many data in the literature suggest that central and peripheral chemoreceptors are involved in the pathogenesis of some SIDS cases. This paper reviews clinical and experimental data indicating that chemoreceptors undergo critical development during the first postnatal period (when the risk of SIDS is higher), are structurally/functionally exposed to most factors triggering SIDS, and may also show structural/functional changes in SIDS victims.

Peripheral chemoreceptors

Postnatal development

The work described here mainly examines the carotid body (CB) as the main peripheral chemoreceptor. However, some authors also believe that other peripheral chemoreceptors, such as aortic bodies, pulmonary neuroendocrine cells and adrenomedullary chromaffin cells, play a role in the pathogenesis of SIDS (Porzionato et al. 2008, 2013).

The CB undergoes structural and functional development during the first postnatal period (the first three postnatal weeks in the rat) (reviewed in Porzionato et al. 2008; De Caro et al. 2013), gradually increasing in hypoxic sensitivity. The period of CB postnatal maturation corresponds to the age range in which the risk of SIDS is highest, indicating that ‘something going wrong’ in this phase may explain at least some cases of SIDS.

Signs of intrinsic vulnerability in SIDS victims

Functional signs

SIDS victims and their siblings have been reported to show prolonged sleep apnoea, excessive periodic breathing, impaired sleep breathing pattern, reduced evoked arousability, and reduced ventilatory sensitivity to hypoxia and hypercapnia (e.g. Valdes‐Dapena, 1980; Slotkin et al. 1995; Gaultier, 2000; Kahn et al. 2003). Such changes may involve peripheral and/or central chemoreceptors. In particular, as regards the arousal response, various central and peripheral components are involved, such as the brainstem respiratory rhythm generator, central chemoreceptors, brainstem and mid‐brain arousal centres, cortically mediated motor responses, and peripheral chemo‐, baro‐ and mechanoreceptors.

In order to evaluate in detail the possible consequences of functional abnormalities, the consequences of CB denervation in the first postnatal period have been studied in several experimental animals. CB denervation within this period causes significant decreases in minute ventilation, respiratory rate, arterial oxygen pressure ($P_{{\mathrm{O}}_2}$) and arousal responses to hypoxia and to water stimulation of the laryngeal reflex, together with increased frequency of apnoeas and higher mortality (e.g. Bowes et al. 1981a, 1981b; Bureau et al. 1985; Grogaard et al. 1986; Fewell et al. 1989; Donnelly & Haddad 1990). These findings support the hypothesis that abnormal or impaired CB development plays a role in the pathogenesis of SIDS.

Structural and neurochemical changes

Obviously, identification of specific pathological findings in the CB of SIDS victims would be of the utmost importance in demonstrating the role played by the CB. Morphometric and neurochemical changes have been reported in the CB of SIDS victims (reviewed in Porzionato et al. 2013). As described in this review, morphometric findings include reduced or increased CB volume, reduced volume and number of glomic cells, higher number of progenitor cells, and reduction and hypoplasia of dense‐cored granules. Conversely, other authors have not found changes in CB volume, mean combined weight, total surface area, functional parenchyma area, number or size of granules, or cytoplasmic argyrophilia in type I cells. Although higher concentrations of dopamine (tenfold) and noradrenaline (threefold) have also been reported in the CB of SIDS victims (Perrin et al. 1984), this finding was not confirmed by another research group (Lack et al. 1986). Some changes are highly intriguing, although at present the lack of recent unbiased studies of confirmation leaves some doubt about the possibility of identifying reliable findings associated with SIDS.

Intrinsic and extrinsic risk factors

Gender and genetic factors

The incidence of SIDS is higher in male than in female infants; conversely, gender and sex steroids are well‐known to influence central and peripheral chemoreception. In the cat, oestrogen and/or progesterone stimulate the responsiveness of hypoxic ventilation and CB neural output. Dopamine and noradrenaline turnover is higher in the CB of female rats; turnover is also increased by orchidectomy and reduced by ovariectomy (Pequignot et al. 1997). Sex‐specific effects on the developmental plasticity of peripheral chemoreception have also been reported. For instance, neonatal exposure to chronic caffeine treatment affects the adult respiratory pattern in male but not female rats, with increased respiratory frequency in the early hypoxic response and increased tidal volume in the late phase (Montandon et al. 2008). High inter‐individual variability has also been reported in the peripheral chemoreceptor function of infants subjected to alternate breath and hyperoxic tests (Bouferrache et al. 2002) and has been ascribed, at least partly, to genetic factors (Thomas et al. 1993; Gaultier & Gallego, 2005). The hypoxic ventilatory response is specifically impaired in newborn mice lacking genes for endothelin‐converting enzyme‐1 (Renolleau et al. 2001) and Nurr1 (Nsegbe et al. 2004).

Prematurity

Prematurity is the main intrinsic factor increasing infant vulnerability to SIDS, and there is much clinical evidence about its detrimental effects on peripheral chemoreception. Premature infants show a higher contribution of peripheral arterial chemoreceptors to baseline ventilation. This parameter is defined by measuring ventilation changes in response to brief hyperoxic stimuli, which rapidly suppress afferents from peripheral arterial chemoreceptors (Gauda et al. 2004). Premature infants have been reported to reduce ventilation by 40% after a brief 100% hyperoxic stimulus during sleep, in comparison with a 14% reduction in full‐term infants (Aizad et al. 1984). The hypersensitivity of peripheral arterial chemoreceptors may precipitate unstable respiration due to apnoeic responses after sighs or brief arousals, and premature infants also show a higher frequency of apnoea. These data support the hypothesis that prematurity may also increase the incidence of SIDS due to its effects on chemoreception. Another intriguing fact is that many conditions associated with prematurity functionally affect chemoreception (see below).

Continuous and/or intermittent hypoxia

The development of the peripheral and central mechanisms of respiratory control may be affected by a series of environmental factors (developmental plasticity), which are known to play a role in SIDS. Premature infants are frequently exposed to hypoxic events, which may further modify the maturation of peripheral arterial chemoreceptors, increasing the risk of disorders of breathing and of SIDS. The literature stresses that various types of hypoxic exposure (continuous or intermittent) may induce differing changes in the structure and function of the CB (Gauda et al. 2004, 2007; Porzionato et al. 2009a).

A reduced ventilatory response to hypoxia has been reported in newborn rats and lambs exposed to chronic continuous hypoxia after birth (e.g. Eden & Hanson, 1987; Sladek et al. 1993) and in infants with bronchopulmonary dysplasia (Calder et al. 1994). Postnatal chronic hypoxia also suppresses the developmental increase of the [Ca²⁺]_i response to hypoxia of rat type I cells (Sterni et al. 1999).

In premature infants, intermittent hypoxia may be more frequent than continuous hypoxia, due to recurrent episodes of apnoea (Peng et al. 2004), and preterm infants with recurrent apnoea show an increased hypoxic ventilatory response, which is correlated with the number of apnoeic episodes (Nock et al. 2004). In preterm infants, the rate of intermittent hypoxia is also correlated with the O₂ saturation target range: preterm infants randomized to a low range (85–89%) have a higher incidence of intermittent hypoxaemia (Di Fiore et al. 2012). In addition, lower 90‐day survival has been reported for infants born small for gestational age with an increased incidence of intermittent hypoxia during the first three postnatal days (Di Fiore et al. 2017).

Postnatal intermittent hypoxia has been reported to increase the hypoxic ventilatory response in piglets (Waters & Tinworth, 2001) and rats (Peng et al. 2004). Significant response differences have also been reported in the exposure of newborn and adult rats to chronic intermittent hypoxia. The increased hypoxic ventilatory response develops with shorter periods of intermittent hypoxia and lasts for longer in newborn than in adult rats (Prabhakar et al. 2007). Thus, a quite convincing body of evidence indicates that prematurity exposes infants to various types of chronic hypoxia (continuous, intermittent, or both) and that these stimuli affect chemoreception.

Hyperoxia

Premature newborns are frequently exposed to supplemental oxygen, which may cause higher peripheral oxygen saturation ($S_{\mathrm{p}{\mathrm{O}}_2}$) levels, and hyperoxaemia may also have a deleterious effect on peripheral chemoreception.

In the rat, various patterns of postnatal hyperoxia exposure have been reported to produce functional changes, such as reduced ventilatory and phrenic nerve hypoxic response (e.g. Ling et al. 1997), reduced single‐unit axonal spiking rates in normoxia and acute hypoxia, lengthening in nerve conduction time (Donnelly et al. 2005), and smaller hypoxia‐induced [Ca²⁺]_i responses (Carroll et al. 2009). Again in the rat, Erickson et al. (1998) reported CB hypoplasia and reduction in the number of carotid sinus nerve axons after one postnatal week of 30% or 60% hyperoxia, and hyperoxia‐induced reduction in CB volume has been reported to persist until adulthood (Bisgard et al. 2003; Bavis et al. 2008).

The probable roles of oxidative stress, functional inactivity, and changes in the expression of trophic factors have been emphasized in hyperoxia‐induced CB hypoplasia. In particular, postnatal hyperoxia exposure upregulates the glomic expression of cerebellin 1 precursor (Dmitrieff et al. 2011) and spexin (Porzionato et al. 2012) in the rat.

However, although there are no doubts about the detrimental effects of hyperoxia on CB development, there is also the fact that hyperoxia is usually far lower in the clinical context than in experimental conditions. In general, it is reasonable to surmise that hypoxic stimuli are more important for maldevelopment and the pathogenesis of SIDS than hyperoxic stimuli.

Exposure to smoke

Maternal cigarette smoking is closely correlated with SIDS. Infants born to mothers who smoke show altered hypoxic arousal (Lewis & Bosque, 1995) and ventilatory responses (Ueda et al. 1999).

Experimental data also show that prenatal and perinatal exposure to nicotine alters the function of the CB. Increased mortality after severe continuous hypoxia has been reported in rats prenatally exposed to nicotine (Slotkin et al. 1995). In newborn rats, prenatal or perinatal nicotine exposure reduces ventilation in normoxia and hypoxia (St‐John & Leiter, 1999) and impairs autoresuscitation after repeated asphyxial stimuli (Fewell & Smith, 1998).

Prenatal (Bamford & Carroll, 1999) or postnatal (Holgert et al. 1995) nicotine administration produces a decreased ventilatory response to hyperoxia in newborn rats. Higher frequencies of spontaneous and post‐hypoxic apnoeas have also been observed in newborn mice (Robinson et al. 2002). In newborn lambs prenatally exposed to nicotine, Hafstrom et al. (2002) observed reduced ventilatory and arousal responses to hypoxia during sleep and a lower ventilatory response to hyperoxia during sleep and wakefulness.

In the carotid bodies at postnatal day (PN) 3 in rats, reduced dopamine and increased expression of tyrosine hydroxylase have been reported after injections of nicotine (Holgert et al. 1995). In the CB of newborn rats, increases in tyrosine hydroxylase and dopamine β‐hydroxylase mRNA have been found after perinatal nicotine exposure (Gauda et al. 2001). Prenatal and early postnatal nicotine exposure also increases the number of cells in the petrosal ganglion expressing tyrosine hydoxylase mRNA at PN3 (Gauda et al. 2001). Prenatal exposure to nicotine also augments mRNA levels of the substance P‐precursor preprotachykinin A in carotid bodies and in the petrosal ganglia of newborn rats at PN1, indicating perturbation of the CB–petrosal ganglion pathway (Berner et al. 2008). The experimental body of evidence of the functional and neurochemical effects of nicotine on the CB is quite sound and consistent, although further confirmation in humans is necessary.

Carbon monoxide (CO) is another constituent of smoke which passes from mother to fetus, leading to tissue hypoxia (Hutter & Blair, 1996). Although CO mainly exerts its effects at the level of the central nervous system, in vitro and in vivo effects on peripheral arterial chemoreceptors have also been reported. Exogenous CO exposure reduces the hypoxic ventilatory response in rats, and this effect has been ascribed to the peripheral chemoreceptors (Zhuang et al. 2006), based upon evidence that CO exposure inhibits CB sensory discharge in hypoxia in the cat (Lahiri et al. 1993) and reverses hypoxic inhibition of the K⁺ channels of CB chemoreceptors in the rat (Riesco‐Fagundo et al. 2001).

Substance abuse

An increased risk of SIDS has been reported in infants perinatally exposed to marijuana (e.g. Scragg et al. 2001) or prenatally exposed to opiates or cocaine during pregnancy (Kandall et al. 1993), although one confounding factor may be maternal cigarette smoking.

Experimental studies have been carried out on the effects of specific substances on chemoreception, although associations in human studies are obviously more difficult to demonstrate.

Cannabinoids have been reported to modulate arousal from sleep in the rat (Carley et al. 2002). In this species, cannabinoid type 1 receptor is expressed in the nodose–petrosal–jugular ganglia complex, superior cervical ganglia and, to a lesser extent, the CB (McLemore et al. 2004).

Infants of opiate‐ or cocaine‐abusing mothers have abnormal sleeping ventilatory patterns (greater duration of apnoea, more periodic breathing) (Ward et al. 1990). Prenatal cocaine blunts the hypoxic response (Lipton et al. 1996a) and decreases the levels of CB dopamine (Lipton et al. 1996b) in PN5 rat pups, and reduces the expression of glial cell line‐derived neurotrophic factor in the CB of rat fetuses at E20.5 (Lipton et al. 1999).

Chronic ethanol exposure is another risk factor for SIDS by reason of studies identifying an association between ‘binge drinking’ during pregnancy and SIDS. Experimental studies in rats and bullfrog tadpoles have identified various effects on breathing, including changes in adaptation to chemosensory challenges (reviewed in Dubois et al. 2013).

Infections and inflammatory events

Infections and inflammatory events are also risk factors for SIDS. One‐third of premature human infants are exposed to intrauterine infection and inflammation. Acute inflammation in newborns is associated with increased frequency and severity of apnoea and intermittent hypoxia (Stock et al. 2010).

As regards the possibility of infective/inflammatory agents exerting their effects through the CB, experimental studies in animals have shown that pre‐ and postnatal lipopolysaccharide (LPS) administration may affect peripheral chemoreception (Fernandez et al. 2008; Samarasinghe et al. 2015; Master et al. 2016). In particular, maternal intraperitoneal LPS administration produces lower baseline ventilation, increased apnoea frequency, and hypersensitivity to hypoxia/hypercapnia in mouse pups (Samarasinghe et al. 2015).

In the rat, intraperitoneal postnatal LPS administration causes more frequent desaturation episodes (hypoxic events, apnoea), attenuated ventilatory responses to changes in oxygen tension, and attenuated hypoxic chemosensitivity of the carotid sinus nerve in in vitro recordings. Carotid bodies show increases in inflammatory cytokines (IL1β, IL6) and the volume fraction occupied by type II cells, together with significant reductions in dopamine content and ultrastructural changes (swelling of mitocondria and Golgi bodies, irregular chromatin condensation) (Master et al. 2016). These experimental studies (although not very numerous) are highly suggestive of the possible detrimental effect of infection/inflammation on ventilatory control through effects on the CB.

Breastfeeding is a well‐known SIDS protective factor and its possible beneficial immunomodulatory role has been proposed. According to the above findings, beneficial anti‐inflammatory and immunomodulatory effects may also be hypothesized in the CB.

Central respiratory chemoreceptors

Postnatal development

The main central respiratory chemoreceptors are considered to be the retrotrapezoid nucleus and subsets of serotoninergic neurons, mainly located in the raphe magnus nucleus. Other structures with possible chemoreceptive functions are some noradrenergic (locus coeruleus) and orexinergic neuronal populations, the ventral medullary surface, and the solitary tract nucleus (Guyenet & Bayliss, 2015). Their differing roles and integration are not yet fully understood. For instance, the various central chemoreceptors may be involved in different ways in sleep and wakefulness (Feldman et al. 2003). Some changes have been reported during development. The parafacial respiratory group has been postulated to be the precursor of retrotrapezoid nucleus in the fetal and early neonatal period (Guyenet et al. 2009).

The CB also projects through the solitary tract nucleus to the retrotrapezoid nucleus (Guyenet & Bayliss, 2015). Inputs from peripheral chemoreceptors can modify the sensitivity of central chemoreceptors, and central stimuli can affect peripheral chemoreceptor sensitivity. Reduced afferent inputs from peripheral arterial chemoreceptors during maturation may also cause maldevelopment of central respiratory centres, leading to further alterations in cardiorespiratory regulation (Gauda et al. 2007).

Newborns are also characterized by immature respiratory control, which includes breathing rhythmicity and its modulation by central/peripheral chemoreceptors and suprapontine influences (Gaultier & Gallego, 2005). Episodes of apnoea (usually central) decrease with advancing postnatal age (Gaultier, 1999) and the hypercapnic ventilatory response increases with postnatal age (Gaultier & Gallego, 2005).

In the rat, although central respiratory centres undergo progressive development during the first two/three postnatal weeks, a specifically critical period has been identified at the end of the second week (PN12–PN13), when a series of neurochemical, metabolic, ventilatory and electrophysiological changes occur (reviewed in Wong‐Riley et al. 2013). During this critical period, the ventilatory response to acute hypoxia is at its weakest, and the expression of tryptophan hydroxylase is also at its lowest in the ventrolateral medullary surface, raphe obscurus and raphe magnus. These changes are particularly suggestive, because the peak incidence of SIDS occurs between the second and fourth months, a period which corresponds to the end of the second postnatal week in the rat in terms of brain development (Wong‐Riley et al. 2013).

Again in the rat, the initial reduction in hypercapnic ventilatory response during the critical period (although not confirmed by all authors) is followed by an increase (Stunden et al. 2001; Wickstrom et al. 2002). In vitro studies have also shown that rat medullary raphe neurons increase their CO₂ chemosensitivity with postnatal age (Richerson, 2004).

In humans, the developmental expression of serotonin receptors has been studied by autoradiography, and shows a progressive reduction in ³H‐LSD binding from fetal to infant to mature age in the nucleus raphe dorsalis, median raphe nucleus and nucleus raphe obscurus (Duncan et al. 2010). ³H‐Nicotine binding also decreases in most brainstem nuclei with increasing age, in both cases of SIDS and controls (Duncan et al. 2008).

On the whole (similar to the above information about peripheral chemoreceptors), the period of critical postnatal development of central respiratory chemoreceptors supports the hypothesis that some structural/functional maldevelopment plays a role in the pathogenesis of SIDS.

Signs of intrinsic vulnerability

According to the above considerations, a series of histopathological, morphometric and neurochemical studies have been performed to examine the possible presence of markers of intrinsic vulnerability in the main respiratory centres. Focusing on central respiratory chemoreceptors, we can refer to the following findings.

Lavezzi et al. (2012) reported hypoplasia or agenesis of the retrotrapezoid nucleus in SIDS and cases of sudden intrauterine death, but some concerns have been raised regarding the true anatomical location of the nucleus in that study (Rudzinski & Kapur, 2013).

Some structures of the medullary serotonin system show significantly lower serotonin 1A receptor binding in SIDS cases than in controls, especially in the paragigantocellularis lateralis and the intermediate reticular zone. The raphe obscurus and paragigantocellularis also show reduced serotonin contents and the raphe obscurus reduced expression of tryptophan hydroxylase (Duncan et al. 2010). Conversely, significant differences in ³H‐nicotine binding between SIDS and controls were not reported in a series of brainstem nuclei, including solitary tract nucleus, locus coeruleus and raphe dorsalis (Duncan et al. 2008).

Another nucleus which has been considered in the past is the arcuate nucleus, because of the presumed homology with the chemosensory area in the ventral medullary surface of lower mammals, such as cats or rodents. In particular, morphometric changes have been reported in SIDS cases, e.g. reduced volume, neuronal density and neuronal (nuclear–cytoplasmic) areas, higher neuronal form factor, and reduced astrocyte density (Matturri et al. 2000; Biondo et al. 2003). Reduced binding to various receptors (muscarinic, kainate, serotoninergic and PACAP receptor 1) has also been reported (Kinney et al. 1995, 2003; Panigrahy et al. 1997, 2000; Huang et al. 2017). In reality, the chemosensory function of the human arcuate nucleus is believed to be speculative by some authors, since it has not yet been demonstrated (Guyenet, 2011).

Reduced tyrosine hydroxylase immunoreactivity and increased numbers of GFAP‐positive glial cells have been reported in the locus coeruleus of SIDS cases. Similar changes have also been described for other structures with noradrenergic neurons, i.e. the area reticularis superficialis ventrolateralis and dorsal vagal nucleus (Obonai et al. 1998).

Although the above morphometric and neurochemical changes further support a pathogenetic role for central respiratory chemoreceptors, they are still difficult to apply for diagnosis in the forensic context, due to the need for specific methodological experience and proper controls for comparison. Of particular interest is a recent paper which reported higher serum serotonin levels in SIDS victims than in controls, with a subset of 31% SIDS cases with serotonin levels higher than 2 standard deviations above the mean of controls, supporting peripheral anomalies in serotonin metabolism and proposing a simple marker (Haynes et al. 2017).

Few studies have examined experimental models of impaired central chemoreception, due to methodological problems, but Cummings et al. (2011) showed that lesions of the lower brainstem serotoninergic neurons compromise autoresuscitation during repeated hypoxia episodes.

Intrinsic and extrinsic risk factors

Gender and genetic factors

Gender effects have also been observed on central respiratory chemoreceptors. Brainstem noradrenergic cell groups (A1, A2, A5) receiving chemosensory fibres show higher dopamine and noradrenaline turnover in female rats; orchidectomy and ovariectomy also respectively increase and reduce dopamine/noradrenaline turnover in these structures (Pequignot et al. 1997). The retrotrapezoid nucleus in female mice shows a higher threshold for c‐fos upregulation in response to CO₂ (10% versus 5%) (Niblock et al. 2012). In the guinea‐pig, oestrogens regulate the neuronal expression of potassium channels contributing to the chemosensitivity of retrotrapezoid cells, such as Kv4.1 (Roepke et al. 2007). The number of potassium channel‐immunoreactive neurons in the medullary raphe also differs between males and females in some inbred rat strains (Riley et al. 2010).

Genetic studies have also reported associations between SIDS and gene variations in the serotonin transporter gene SLC6A4 (e.g. Weese‐Mayer et al. 2003; although their results were not confirmed by Paterson et al. 2010) and serotonin 1A receptor gene HTR1A (Morley et al. 2008), with possible implications in central chemoreception on the part of the serotoninergic system.

Genetic factors are also probably involved in inter‐individual variability in hypercapnic ventilatory responses (Gaultier & Gallego, 2005). In inbred adult mouse strains, the hypercapnic ventilatory response is genetically determined (Tankersley, 2003). In addition, an abnormal hypercapnic ventilatory response has been reported in mice lacking specific genes such as Htr1a and the Mash‐1‐Ret‐Phox2b signalling pathways as well as genes for endothelin‐1 and PACAP (e.g. Burton et al. 1997; Kuwaki et al. 1999; Cummings et al. 2004; Baccini et al. 2012).

Prematurity

Clinical and experimental data indicate reduced central chemoreception in prematurity. Preterm newborns show breathing irregularity, periodic breathing, frequent episodes of apnoea and weaker hypercapnic ventilatory response (Zhang et al. 2003; Darnall, 2010). The incidence of periodic breathing and apnoea decreases from 30 to 40 weeks post‐menstrual age (Parmelee et al. 1972). Periodic breathing has been correlated to a decrease in ventilation and CO₂ sensitivity (Rigatto & Brady, 1972), with the apnoeic threshold closer to eupnoic CO₂ levels (Khan et al. 2005).

Hypoxic and hyperoxic stimuli

Hypoxia and hyperoxia mainly affect the developmental plasticity of peripheral arterial chemoreception, although some studies also report a few minor central effects. For instance, in an integration of experimental data and a computational model, Molkov et al. (2011) proposed that chronic intermittent hypoxia increases the CO₂ sensitivity of the retrotrapezoid nucleus/parafacial respiratory group in the rat.

In the rat, exposure to sustained hypoxia at PN11–PN15 (critical period) reduces the ventilatory response to acute hypoxia and increases mortality (Mayer et al. 2014; MacFarlane et al. 2016). Although a possible role played by the CB cannot be excluded, increased numbers of microglial cells and decreased serotonin immunoreactivity have been reported in the solitary tract nucleus and dorsal vagal nucleus. The above changes can be prevented by minocycline treatment, thus supporting the role played by microglial cells (MacFarlane et al. 2016).

Perinatal hyperoxial exposure of rat pups has also been reported to reduce the frequency of phrenic bursting generated by the central respiratory network, at baseline and in response to hypoxia (Bierman et al. 2014).

Exposure to smoke

Many experimental studies have shown that prenatal/perinatal exposure to nicotine affects the postnatal development of central breathing patterns and chemoreception. In the rat, prenatal nicotine exposure is correlated with a higher incidence of apnoea in the first two postnatal days, and higher breathing frequency and lower tidal volume in later periods, together with blunting of ventilatory responses to hypercapnia and/or hypoxia (Huang et al. 2004, 2010). In the mouse, prenatal to early postnatal nicotine exposure reduces basal minute volume and ventilatory responses to hypercarbia and hypoxia in the first postnatal days (Eugenin et al. 2008). In the same study, isolated brainstem–spinal cord preparations from PN0–PN3 nicotine‐exposed newborns showed longer and more irregular fictive respiratory cycles, together with a lower response to acidification. A switch from muscarinic to nicotinic receptor‐based mechanisms has also been reported in the cholinergic component of central respiratory chemoreception (Eugenin et al. 2008). Lei et al. (2015) report that, in medullary slices sampled from prenatally exposed rats, responses to acidification (increased burst frequency and reduced integrated amplitude) are lower than in controls, indicating a blunting effect on central chemoreception. In mouse, prenatal to early postnatal nicotine exposure also causes specific changes in the raphe obscurus nucleus (decreased number of serotoninergic neurons and increased expression of 5‐HT_1A autoreceptors) (Cerpa et al. 2015).

Reduced serotoninergic binding has been reported in the arcuate nucleus of infants exposed to perinatal maternal smoking (Kinney et al. 2003). Reduced ³H‐nicotinic receptor binding in the locus coeruleus, periacqueductal grey matter and raphe dorsalis has been reported in control cases exposed to maternal smoking in utero with respect to other controls, although similar differences were not found in SIDS cases (Duncan et al. 2008).

As regards other constituents of cigarette smoke, newborn guinea‐pigs prenatally exposed to CO have been reported to show significantly greater tidal volume and minute ventilation during steady‐state hypercapnia, indicating increased sensitivity to CO₂ (McGregor et al. 1998). Newborn guinea‐pigs prenatally exposed to CO also showed a significant decrease in tyrosine hydroxylase expression in the solitary tract nucleus, dorsal vagal nucleus, area postrema, intermediate reticular nucleus and ventrolateral medulla, and significantly increased choline acetyltransferase expression in the dorsal vagal nucleus and hypoglossal nucleus (Tolcos et al. 2000).

Infections and inflammatory events

Infections and inflammatory events may also have negative effects on chemoreception and respiratory control through central mechanisms, as attenuated hypoxic ventilatory responses following intratracheal LPS have also been reported in the rat after carotid sinus nerve transection (Balan et al. 2011). In addition, vulnerability to LPS is highest in the critical period for the development of central respiratory centres. In the rat, intraperitoneal injection of LPS attenuates the early and late phases of the acute hypoxia ventilatory response if performed at PN10, but not at PN5 or PN20; increased mortality and increased expression of TNFα and iNOS in the brainstem have also been reported (Rourke et al. 2016).

Methodological considerations and future perspectives

In conclusion, the studies reviewed here strongly support the role of central and peripheral respiratory chemoreceptors in the pathogenesis of SIDS. It has been amply demonstrated that respiratory chemoreceptors undergo critical development during the postnatal period, when SIDS typically occurs, and that this development may be structurally/functionally affected by most risk factors for SIDS. In spite of this, conclusive demonstration of chemoreception impairment in SIDS cases is still difficult, due to a series of methodological problems concerning human material.

As regards studies on autopsy material, the main problem is difficulty in obtaining large standardized series of cases and controls. Particularly in a rare and (probably) heterogeneous entity such as SIDS, reliable results can only derive from large and highly standardized series. In case‐control studies, we know that many confounding factors may bias the statistical results. In the available literature on the matter, possible biases due to under‐matching, over‐matching or other mismatching of controls are not always adequately considered. Higher attention should be paid in sampling controls and justifying the case:control ratio.

Studies on morphometric parameters in the comparative analysis of central and peripheral chemoreceptors between SIDS victims and controls are described here. However, most of these approaches show methodological problems, usually involving parameters calculated on two‐dimensional planes, such as surface areas or cell densities per surface area. Counting on a two‐dimensional plane produces intrinsic bias in estimating cell numbers, because the probability of objects being hit by a single section is proportional to their size and form, and does not depend only on their number. Thus, stereological methods should be applied, with three‐dimensional probes (disectors or fractionators) which permit sampling of objects with a probability which is proportional only to their number (West, 1993). Some authors have reported differences of as much as 40% between two‐dimensional (biased) methods and three‐dimensional (unbiased) methods (Pakkenberg et al. 1991; Coggeshall, 1992). Obviously, design and measurement take precedence over statistics and biased measures cannot be rescued by statistical analysis. In a previous study, we performed stereological analysis of the main medullary nuclei of adults and infants according to the optical dissector method. The infant series contained both cases of SIDS and controls, but significant differences were not found, although this may have been due to the small number of cases (Porzionato et al. 2009b). Further unbiased stereological studies will be necessary to verify possible morphometric changes in central and peripheral respiratory chemoreceptors of SIDS victims.

One of the main problems of working with human postmortem material is tissue degradation, which may affect results in unpredictable ways and which is difficult to standardize between cases and controls.

The above problems have particularly affected research on the CB, as this structure is still rarely sampled in SIDS autopsies. Standardized collection of the CB in SIDS would be necessary to develop large tissue banks for analysis, but international standardized autopsy protocols for SIDS do not usually include CB sampling. For instance, some intriguing aspects of the CB in SIDS have not yet been investigated, e.g. the expression of several neurotransmitters/neuromodulators and their corresponding receptors and transporters. Nevertheless, findings on experimental animals clearly indicate modulation of neurotransmission/neuromodulation in response to SIDS risk factors.

Apart from autopsy studies, further functional studies on prematures and full‐term newborns would be useful to identify infants at risk of SIDS. In fact, most experimental studies on animals have identified functional (rather than structural) effects as the result of intrinsic/extrinsic risk factors for SIDS. Prospective studies addressing some simple functional tests on large infant series would be the only way of specifically identifying and correlating central and/or peripheral chemoreceptor impairment with the risk of SIDS. Some authors have also suggested evaluation of chemoreceptor function (hyperoxia and alternate breath tests) in preterm infants before hospital discharge (Gaultier & Gallego, 2005). Genetic factors have also been mentioned as playing a role in breathing instability; thus, integration of functional and genetic data in prospective clinical studies would be the gold standard. Obviously, the design of such studies would need consideration of technical, clinical and ethical issues of particular importance and difficulty.

Additional information

Competing interests

None of the authors has any conflict of interests.

Author contributions

All the authors have contributed to writing the paper. All authors approved the final version of the manuscript. All persons designated as authors qualify for authorship and all those who qualify for authorship are listed.

Funding

This work was funded through a Research Project of the University of Padova (PRAT 2014 Andrea Porzionato: CPDA141382/14).

Biographies

Andrea Porzionato is a specialist in legal medicine and achieved a PhD in neuroscience.

Veronica Macchi specialized in radiology and achieved PhD in neuroscience.

Raffaele De Caro specialized in pathological anatomy and legal medicine. All the authors are Professors of Human Anatomy in the University of Padua and they are involved in judicial autopsies. Professor De Caro is the Director of the Institute of Human Anatomy, Past‐President of the European Association of Clinical Anatomy and President of the Italian College of Human Anatomists. The research interests of the group include the central and peripheral structures of respiratory control, investigated through histopathological and morphometric/stereological approaches in human autoptic material (e.g. drug‐related deaths, sudden infant death syndrome, abusive head trauma) and experimental animal studies (e.g. exposure to hypoxic, hyperoxic or inflammatory stimuli).