Acta Neuropathol DOI 10.1007/s00401-015-1437-9

ORIGINAL PAPER

Decreased orexin (hypocretin) immunoreactivity in the hypothalamus and pontine nuclei in sudden infant death syndrome Nicholas J. Hunt1,2 · Karen A. Waters1,2,3 · Michael L. Rodriguez4,5 · Rita Machaalani1,2,3 

Received: 14 January 2015 / Revised: 28 April 2015 / Accepted: 29 April 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Infants at risk of sudden infant death syndrome (SIDS) have been shown to have dysfunctional sleep and poor arousal thresholds. In animal studies, both these attributes have been linked to impaired signalling of the neuropeptide orexin. This study examined the immunoreactivity of orexin (OxA and OxB) in the tuberal hypothalamus (n  = 27) and the pons (n  = 15) of infants (1–10 months) who died from SIDS compared to age-matched non-SIDS infants. The percentage of orexin immunoreactive neurons and the total number of neurons were quantified in the dorsomedial, perifornical and lateral hypothalamus at three levels of the tuberal hypothalamus. In the pons, the area of orexin immunoreactive fibres were quantified in the locus coeruleus (LC), dorsal raphe (DR), laterodorsal tegmental (LDT), medial parabrachial, dorsal tegmental (DTg) and pontine nuclei (Pn) using automated

Electronic supplementary material  The online version of this article (doi:10.1007/s00401-015-1437-9) contains supplementary material, which is available to authorized users. * Rita Machaalani [email protected] 1

Department of Medicine, Room 206, SIDS and Sleep Apnoea Laboratory, Sydney Medical School, University of Sydney, Blackburn Building, D06, Sydney, NSW 2006, Australia

2

BOSCH Institute of Biomedical Research, University of Sydney, Sydney, NSW, Australia

3

The Children’s Hospital, Westmead, NSW, Australia

4

Department of Forensic Medicine, NSW Forensic and Analytical Science Service, Sydney, NSW, Australia

5

Department of Pathology, Sydney Medical School, The University of Sydney, Sydney, NSW, Australia





methods. OxA and OxB were co-expressed in all hypothalamic and pontine nuclei examined. In SIDS infants, orexin immunoreactivity was decreased by up to 21 % within each of the three levels of the hypothalamus compared to non-SIDS (p  ≤ 0.050). In the pons, a 40–50 % decrease in OxA occurred in the all pontine nuclei, while a similar decrease in OxB immunoreactivity was observed in the LC, LDT, DTg and Pn (p ≤ 0.025). No correlations were found between the decreased orexin immunoreactivity and previously identified risk factors for SIDS, including prone sleeping position and cigarette smoke exposure. This finding of reduced orexin immunoreactivity in SIDS infants may be associated with sleep dysfunction and impaired arousal. Keywords  Human · SIDS · Development · Sleep · Sleep dysfunction · REM · Automated fibre quantification Abbreviations DMH Dorsomedial hypothalamus DR Dorsal raphe DTg Dorsal tegmental nucleus LH Lateral hypothalamus LDT Laterodorsal tegmental nucleus LC Locus coeruleus MPB Medial parabrachial nucleus Ox Orexin OxA Orexin A OxB Orexin B PPT Pedunculopontine tegmental area PeF Perifornical area Pn Pontine nucleus PPO Prepro-orexin REM Rapid eye movement URTIs Upper respiratory tract infections

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Background Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal (1–12 months of age) death in Australia and the developed world. SIDS is defined as the sudden death of an infant less than 1 year of age that cannot be explained after a thorough investigation is conducted, including a complete autopsy, examination of the death scene and a review of the clinical history [28]. SIDS, therefore, is a diagnosis of exclusion. SIDS mostly occurs during sleep, leading to the proposal that SIDS infants have dysfunctional maturation of sleep- and respiratory-related control [97]. In support of this hypothesis, multiple neurochemical abnormalities have been found in SIDS victims, in areas associated with sleep regulation, arousal and physiological responses to hypoxic and/or hypercapnic stressors [17, 27, 54, 65, 81, 82]. Hypoxia (and/or hypercapnia) has been shown to be associated with co-sleeping or a prone sleeping position; known risk factors for SIDS [43, 48]. Orexin (Ox), also called hypocretin, refers to a pair of neuropeptides (orexin A [OxA] and orexin B [OxB]) expressed within neurons in the dorsomedial, perifornical and lateral hypothalamus (DMH, PeF and LH, respectively) [21, 45, 83]. OxA and OxB are produced from the same precursor protein prepro-orexin (PPO) [83] and packaged into vesicles for axonal transport [85]. Orexinergic axons are found throughout the CNS except the cerebellum [76]. Ox is critical for maintaining wakeful/ sleeping states, levels of arousal, stabilisation of rapid eye movement (REM) and non-REM sleep, and contributes to maintaining stable (non-apnoea) respiration during sleep [30, 84] via connections with monoaminergic and cholinergic neurons in the pons (locus coeruleus [LC], dorsal raphe nucleus [DR], pontine tegmentum [pedunculopontine tegmental area [PPT] and laterodorsal tegmental nucleus [LDT]]) and the tuberal mammillary nucleus in the hypothalamus [84]. Neurons in these areas are critical for the regulation and control of REM and non-REM sleep, and, in part, for induction of arousal from sleep under both basal and stressed (e.g. asphyxial) conditions [38, 62]. A >95 % reduction of Ox immunoreactivity in the hypothalamus due to loss of Ox neurons in mice and humans produces the sleep disorder narcolepsy [11, 99]. Moderate (25–35 %) reductions in Ox immunoreactivity induce impaired sleep regulation, particularly REM sleep [14]. Furthermore, Ox regulates energy metabolism and the sympathetic stress response [84]. During stress, basal levels of Ox are elevated to promote and consolidate arousal [84]. Finally, orexinergic stimulation promotes neuroprotection and survival [108]. Given that SIDS infants demonstrate impaired sleep regulation [47, 52], in this study it was hypothesised that they may have a decrease in Ox levels, similar to

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Acta Neuropathol

that demonstrated in hypoxia-exposed animal models of impaired sleep regulation [61]. Using piglets exposed to hypercapnic hypoxia, we found reduced Ox levels in the hypothalamus [26] and in other animal studies, hypoxic or hypercapnic insults result in decreased PPO levels in the hypothalamus of similar magnitude to those associated with impaired REM sleep in mice [61, 101]. It was, therefore, hypothesised that SIDS infants may have a decrease in Ox immunoreactivity in both the hypothalamus and pons: possibility mediated by exposure to hypoxia and/or hypercapnia as may occur when sleeping in the prone position. However, since hypoxia/reoxygenation injury in mice causes reduced PPO synthesis without Ox neuron apoptosis [109], in SIDS infants, the total number of hypothalamic Ox neurons may not change. Using immunohistochemistry, we tested these hypotheses by examining OxA and OxB immunoreactivity in the hypothalamus and the pons and counted the number of neurons present in the hypothalamus in SIDS infants compared with age-matched nonSIDS infants.

Materials and methods Tissue collection and hypothalamus localization Seven-micron sections mounted on 3-aminopronopyltriethoxysilane-coated slides, of formalin-fixed, paraffin-embedded tissue from the hypothalamus and the rostral pons were obtained from SIDS (n  = 27) and age/sex matched non-SIDS (n = 19) infants from the Department of Forensic Medicine (Sydney, NSW, Australia). The cause of death for the non-SIDS infants included septicaemia (n = 3), gastroenteritis (n = 2), bronchopneumonia (n = 3), myocarditis (n = 3), asphyxia (n = 3), cardiac failure (n = 2), and sudden accidental death (n  = 3). For each case, sections from 1 (n = 8), 2 (n = 34) or 3 (n = 4) levels of the hypothalamus were available. The three levels of the hypothalamus (anterior, central and posterior tuberal hypothalamus; Fig. 1), correspond to Figs. 26, 27, 28 in Mai et al. [67] and Fig. 1E, F, G–H of [96]. Since the density of Ox neurons in the hypothalamus varies depending on the anatomical level [29], only sections corresponding to these anatomically defined levels were included. Each level contained the fornix at a different location relative to hypothalamic nuclei in the hypothalamus. The fornix descends towards the mammillary body at 3 mm/mm3 in the adult human tuberal hypothalamus [67]. In infants this rate is 1.5–2 mm/mm3 [96]. Each anatomical level (anterior, central and posterior) is separated by 1 mm corresponding to a 1.5–2 mm change in the position of the fornix in the sections as shown in Fig. 1. Sections from the rostral pons were available for 15/27 SIDS and 8/19 non-SIDS cases. These were taken +26 or

Acta Neuropathol Fig. 1  Schematic drawings and Nissl stained sections of the anterior, central and posterior tuberal hypothalamus. ARC arcuate nucleus, DMH dorsal medial hypothalamus, f fornix, LH lateral hypothalamus, LTu lateral tuberal area, PeF perifornical area, PVN paraventicular nucleus, SON supraoptic nucleus, TM tuberal mammillary, VMH ventral medial hypothalamus. Scale bar 1000 µm. Levels were determined with reference to figures 26, 27 and 28 from the Atlas of the Human Brain [67]

+27 mm rostral to the obex and correspond to Figs. 43 and 44 in Paxino and Huang [80]. The LC, DR, LDT, medial parabrachial nucleus (MPB), dorsal tegmental nucleus (DTg) and the pontine nucleus (Pn) were identified with reference to Paxino and Huang [80, Fig. 1G, H]. Data relating to the infant cases was obtained from police reports, including the P534 NSW Police Death Scene Investigation checklist for Sudden Unexpected Death in Infancy (including SIDS), and pathology reports and included sex, age at death, post conceptional age, cause of death, body weight, body length, head circumference, the presence of SIDS risk factors [prone sleeping position, cigarette smoke exposure, recent upper respiratory tract infections (URTIs) and prematurity], post-mortem interval, pulmonary integrity, the presence of petechiae, brain weight, duration of formalin fixation and the presence of gliosis. Data regarding sleep quality, appetite and metabolic state were not available. This study was approved by the Human Research Committees of the University of Sydney, the Sydney Local Health District (Royal Prince Alfred Hospital Zone) and the Office of the NSW Coroner. For this anonymized retrospective study, formal consent is not required. Immunohistochemistry Immunohistochemistry was performed as previously reported [45] using anti-OxA (sc-8070; Santa Cruz

Biotechnology Inc., USA) and anti-OxB (sc-8071; Santa Cruz Biotechnology Inc., USA) primary antibodies raised against Ox peptides. These antibodies have been extensively characterised as summarised in Supplementary Table 1 [45]. All steps were carried out at room temperature, unless otherwise stated. Briefly, formalin-fixed paraffin embedded sections were deparaffinised and antigen retrieved by microwaving in TRIS–EDTA buffer (1 mM EDTA, 1 mM trisodium citrate, 2 mM TRIS; pH 9.0) for 14 min. Endogenous peroxidase was quenched using 3 % H2O2, 50 % methanol in PBS for 20 min. After blocking in 10 % normal horse serum for 1 h, sections were incubated overnight with the respective primary antibody; sc-8070 (1:1000), sc-8071 (1:1000). Sections were then incubated with biotinylated horse anti-goat secondary antibody (1:400, BA-9500, Vector Laboratories, CA, USA) for 1 h, followed by avidin–biotin complex (VEDH-4000, Vector Laboratories, CA, USA) for 1 h. Colour labelling was developed with 3,3′-diaminobenzidine (VESK-3100, Vector Laboratories, CA, USA). Hypothalamic sections were lightly counterstained with Harris’ haematoxylin; pontine sections were not counterstained. Sections where then dehydrated and mounted in DPX. For negative controls, the primary antibody was replaced with 1 % normal horse serum. Duplicate staining was performed to confirm both reproducibility of staining and quantification.

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Quantification and statistical analysis In the hypothalamus, the border between the lateral edge of the DMH and the medial edge of the PeF is poorly defined histologically [45]. We arbitrarily defined the lateral edge of the DMH to be 2/3 of the distance from the 3rd ventricle to the middle of the fornix [45] (Fig. 1). The remaining borders of the DMH, PeF and LH were histologically well-defined. The total number of neurons and the number of Ox immunoreactive neurons were manually counted in the DMH, PeF and LH at the anterior (non-SIDS n = 5, SIDS n = 5) and central (non-SIDS n = 9, SIDS n = 14) tuberal levels and in the DMH and LH at the posterior tuberal level (non-SIDS n = 5, SIDS n = 9) using a counting grid, and the percentage of Ox immunoreactive neurons in each region and in all three regions combined, was calculated [45]. The number of Ox immunoreactive neurons per section was compared with those reported by Fronczek et al. [29] who used an unbiased stereological method. This group provides the only quantitative data on the number of Ox neurons at each level in the human hypothalamus [26]. All counting was performed blinded to case identification. For neuronal counting, sections were examined at 10× magnification with a Leica upright DM6000B microscope (Nikon Corporation, Tokyo, Japan) and images of the three regions were captured using image capture software (LAS V4.2, Leica Microsystems Ltd. Heerbrugg, Switzerland). Only neurons with a clearly defined nucleolus were counted. The total number of Ox immunoreactive neurons at each level was determined by summing the neuronal counts from the DMH, PeF and LH. Neuronal size and the presence of coarse granular Ox immunoreactive particles (indicative of clumping of the neuropeptides) were determined using image capture software (LAS V4.2, Leica Microsystems Ltd. Heerbrugg, Switzerland). In the pons, the area of OxA and OxB immunoreactivity fibre expression were quantified using the methods of Grider et al. [32] and Sathyanesan et al. [88]. Sections were examined at 20× magnification and images were captured using a Leica upright DM6000B microscope (Nikon Corporation, Tokyo, Japan) and image capture software (LAS V4.2, Leica Microsystems Ltd. Heerbrugg, Switzerland). Images were opened in ImageJ (National Institute of Health, USA) (Fig. 2c), the background was subtracted and the image inverted to produce bright OxA or OxB immunoreactive fibre staining on a dark background. A smallest absolute Eigen value Hessian filter (smoothing scale 1.0) was applied (Fig. 2d). Following this the image was converted to a binary image using a ‘triangle’ threshold in ImageJ (Fig. 2e). Raw data were obtained by automated thresholding using the ‘analysis particles’ function in ImageJ with the set measurements giving the number of

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Acta Neuropathol

pixels/mm2 and the percentage of the image area occupied by OxA or OxB immunoreactive fibres. To ensure that the automated thresholding was uniform and did not produce error, manual thresholding of all non-SIDS pontine sections was performed. Thresholding by either method produced similar results (p = 0.98; Fig. 2f). Data represent the area of fibre immunoreactivity/mm2 (pixels/mm2). All raw data were imported into Microsoft Excel 2010 (Microsoft Windows Inc., Washington, USA); the mean  ± SEM percentage of Ox immunoreactive neurons for each region (hypothalamus) and the mean area of fibre immunoreactivity  ± SD for each nucleus (pons) for nonSIDS and SIDS cases were calculated. The percentage of Ox immunoreactive neurons in each region was determined by dividing the number of Ox immunoreactive neurons by the total number of neurons counted. Normal distribution was examined using the Shapiro–Wilk test given our low n value. The results for non-SIDS and SIDS groups were compared using one-way ANOVA in SPSS for Windows [V21; SPSS (IBM) Inc., Illinois, USA]. Due to the limited number of sections from the anterior tuberal level (n  = 5, non-SIDS; n  = 5, SIDS) post hoc Kruskal–Wallis one-way analyses were performed to determine if the observed differences were significant assuming a nonnormal distribution. Correlated bivariate Pearson’s analysis was undertaken within the SIDS group to identify possible associations between changes in Ox immunoreactivity in the hypothalamus and pons and SIDS risk factors. Post hoc linear regression was performed to determine the relationship between Ox immunoreactivity in the hypothalamus and the pons.

Results Non-SIDS and SIDS groups were well matched for demographic features including sex, age at death, body weight and length, brain weight and head circumference, and for parameters that may affect immunohistochemical staining, including post-mortem interval and duration of tissue fixation (Table 1). As expected, known risk factors for SIDS prone sleeping [55 vs 31 %; p = 0.05] and cigarette smoke exposure [56 vs 29 %; p = 0.05] were more common than in non-SIDS cases, while other risk factors such as URTIs and prematurity were not significantly different (Table 1). Intrathoracic petechiae, pulmonary congestion and oedema and aspiration of stomach contents were more common in SIDS cases (p ≤ 0.05; Table 1). In the hypothalamus, diffuse cytoplasmic OxA and OxB immunoreactivity was seen in round, elliptical or fusiform neurons between 20 and 50 µm in maximum diameter. There was no significant difference in neuronal diameter between non-SIDS and SIDS infants (p  = 0.91; Fig. 3).

Acta Neuropathol Fig. 2  Development of binary OxA immunoreactive fibre stained images and comparison of manual and automated thresholding; comparison between non-SIDS and SIDS fibre immunoreactivity in the LC. Non-SIDS (a) and SIDS (b) fibre immunoreactivity in the LC. The raw image of OxA immunoreactive staining of the LDT (c) is converted with an absolute smallest Eigen value hessian filter (d), followed by automated thresholding and binary functions (e). Manual and automated thresholding methods are compared in the non-SIDS group (n = 9; f). Data shows the mean ± SD in area of immunoreactivity (pixels/mm2). Black arrows indicate examples of positive immunoreactivity within fibre projections. Scale bar 100 μm

Coarse granular Ox immunoreactivity, corresponding to the Ox aggregates described in murine apoptotic neurons, was not identified [67] (Fig. 3). Based on a review of the pathology reports, no gliosis was identified in the hypothalamus or the pons in either SIDS or non-SIDS infants. In non-SIDS infants, the total number of Ox immunoreactive neurons in a 7 µm section within the anterior, central and posterior tuberal hypothalamus was 14 % greater than that observed by Fronczek et al. [29]; this study used stereological methods and described the same three areas used in our study. The difference in Ox neuronal numbers is likely due to age-related changes since infant Ox neuronal numbers are 13 % greater in infants compared to adults [45]. The ratio of Ox neuronal numbers in the 7-µm section between the three sections (anterior-central 0.28; anteriorposterior 0.75; central-posterior 2.52) was similar to that reported by [29] (0.26; 0.72; 2.69, respectively). Compared to non-SIDS cases, there was a significant decrease in the percentage of Ox immunoreactive neurons in the tuberal hypothalamus in SIDS cases, at each of

the three levels examined: a 19 % decrease in the anterior (p  = 0.033), an 11 % decrease in the central (p  = 0.001) and a 21 % decrease in the posterior (p  = 0.005) tuberal hypothalamus (Fig. 4). However, when individual subareas were compared, a significant difference was only identified in the LH of the posterior tuberal hypothalamus (p  = 0.04, Table 2). The significant decreases observed when each level was considered as a whole most likely reflect the effects of summation of the smaller non-significant decreases noted in the other sub-regions (Table 2). Regarding the total neuron numbers (total of orexin and non-orexin neurons), there was no difference between nonSIDS and SIDS cases at any level or within any region (Fig. 5). In the pons, no significant differences were observed in the area of immunoreactive fibre staining between the two neuropeptides (OxA and OxB) in the pontine nuclei in nonSIDS infants (p  ≥ 0.53; Figs. 4, 6). In the pons, the LC had the greatest Ox immunoreactivity while the Pn had the least (Fig. 6). Compared to non-SIDS infants, there was a

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Table 1  Non-SIDS and SIDS infant dataset

Acta Neuropathol Characteristics

Control (n = 19)

SIDS (n = 27)

Male: female Age of death (months) Post conception age at death (months) Weight (kg) Length (cm) Brain weight (kg) Head circumference (cm) Risk factors  Prone sleep position  Smoke exposure  URTI  Preterm Autopsy/tissue related factors  Pulmonary oedema/aspiration/congestion  Petechiae: thymic/lungs/epicardial  Post mortem interval (h)

11:8 5.6 ± 3.1 14.5 ± 3.2 7.0 ± 2.5 63.8 ± 8.3 0.8 ± 0.2 42.4 ± 4.8

15:12 4.9 ± 2.8 13.9 ± 2.9 6.8 ± 1.9 62.2 ± 8.9 0.8 ± 0.2 41.5 ± 3.5

0.49 0.54 0.59 0.80 0.60 0.56 0.53

31 % 29 % 38 % 38 %

55 % 56 % 46 % 16 %

0.05 0.05 0.44 0.26

23 %, 0 %, 46 % 0 %, 0 %, 5 % 35.4 ± 25.7

40 %, 15 %, 62 % 59 %, 41 %, 41 % 27.6 ± 17.7

7.0 ± 2.8

6.3 ± 3.5

 Fixation duration (weeks)

p value

≤0.05 ≤0.05 0.27 0.41

Non-SIDS causes of death: septicaemia (n = 3), gastroenteritis (n = 2), bronchopneumonia (n = 3), myocarditis (n = 3), asphyxia (n = 3), cardiac failure (n = 2), sudden accidental death (n = 3) Bold numbers indicate the significant values Fig. 3  OxA and OxB immunoreactive neurons in (a, c) non-SIDS and (b, d) SIDS PeF. Scale bar 50 μm

40–50 % decrease in OxA fibre density in SIDS infants in all pontine nuclei examined (p ≤ 0.004). A similar decrease (40–50 % decrease) in OxB fibre density was observed in the LC, LDT, DTg and Pn (p ≤ 0.025, Fig. 6). There was no association between decreased Ox immunoreactivity in the hypothalamus or the pons and risk factors for SIDS or pathological features identified at post

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mortem (p  ≥ 0.32; Table 3). No linear correlation was observed with post conceptional age (p  = 0.92). In SIDS cases, a linear relationship exists between the percentage of reduced Ox immunoreactivity in the hypothalamus and the LC (Fig. 7a) and MBP pontine nuclei (p = 0.032, p = 0.016). Indicating expression changes in the hypothalamus relate to changes in fibre area expression in the pons.

Acta Neuropathol

Fig. 4  Box and whisker plot comparison of the percentage OxA and OxB immunoreactive neurons in non-SIDS and SIDS infants. Data show the total percentage of OxA and OxB neurons in the tuberal hypothalamus at the anterior (non-SIDS n  = 5, SIDS n  = 5), cen-

tral (non-SIDS n = 9, SIDS n = 14) and posterior (non-SIDS n = 5, SIDS n  = 8) levels with error bars representing range. *p  ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

Fig. 5  Total neuron numbers in the three levels of the hypothalamus in non-SIDS and SIDS infants. Comparison between non-SIDS (white) and SIDS (black) in the anterior (a; non-SIDS n  = 5, SIDS

n  = 5), central (b; n  = 9, SIDS n  = 14) and posterior (c; nonSIDS n = 5, SIDS n = 8) tubular hypothalamus. Data represent the mean ± the SEM

Fig. 6  Quantifiable fibre immunoreactivity of OxA and OxB in non-SIDS and SIDS infants. Comparison between non-SIDS (white; n = 8) and SIDS (black; n = 15) for OxA immunoreactive (a) and OxB immunoreactive (b) expression in the DR, LC, LDT, MPB, DTg and Pn. Data represent the mean ± the SD of pixels/mm2. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

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Table 2  Comparison of the percentage OxA and OxB immunoreactive neurons in nonSIDS and SIDS infants

Acta Neuropathol Nuclei

Anterior  DMH  PeF  LH Central  DMH  PeF  LH Posterior  DMH  LH

OxA

OxB

Non-SIDS

SIDS

p value

Non-SIDS

SIDS

p value

6.58 ± 0.57 13.28 ± 1.26 8.70 ± 1.03

6.01 ± 0.69 11.13 ± 0.24 5.26 ± 1.23

0.56 0.10 0.08

6.58 ± 0.57 13.28 ± 1.26 8.70 ± 1.03

6.67 ± 0.70 10.62 ± 0.47 6.06 ± 1.17

0.92 0.07 0.14

20.12 ± 1.20 33.81 ± 1.37 26.07 ± 1.62

21.28 ± 0.74 32.68 ± 0.87 22.02 ± 1.50

0.41 0.48 0.09

19.88 ± 1.27 33.51 ± 1.42 25.78 ± 1.69

21.41 ± 0.75 33.31 ± 1.00 21.60 ± 1.50

0.30 0.91 0.08

10.18 ± 1.04

9.56 ± 0.44

0.58

10.42 ± 1.06

10.10 ± 0.50

0.76

15.91 ± 0.55

12.15 ± 0.96

0.04

15.53 ± 0.54

12.85 ± 0.61

0.43

Data show the DMH, PeF and LH percentage of Ox neurons in the anterior (non-SIDS n = 5, SIDS n = 5), central (non-SIDS n = 9, SIDS n = 14) and posterior (non-SIDS n = 5, SIDS n = 8) tuberal hypothalamus. Data represent the mean ± the SEM Bold number indicates the significant value

Fig. 7  Ox immunoreactivity in the tuberal hypothalamus compared to the LC pons; and the small and large reductions in Ox immunoreactivity in SIDS infants. a Linear relationship between decreased Ox immunoreactivity (normalised to non-SIDSs mean) in the tuberal hypothalamus and the LC pons in SIDS infants (line of best fit and 95 % conference intervals shown). b Comparison of the percentage

of Ox immunoreactivity (normalised to non-SIDS mean) between non-SIDS (HT: n  = 19; pons: n  = 8) and the small (HT: n  = 18; pons: n = 9) and large (HT: n = 9; pons n = 6) reduction groups in SIDS. Data represent the mean ± the SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001

The decrease in Ox immunoreactivity in the tuberal hypothalamus varied between individual SIDS cases (5–30 %; Fig. 7a). For both the total tuberal hypothalamus and the LC of the pons, there appeared to be two groups of SIDS cases, with either “small” (hypothalamus: n = 18/27, LC: 9/15) or “large” (hypothalamus: n  = 9/27; LC: 6/15) reductions in immunoreactivity relative to the non-SIDS. No SIDS risk factor or pathological features were identified using covariate analysis with either the small or large change in expression cases (p  ≥ 0.138). Shapiro–Wilk analysis demonstrates that SIDS cases had a non-normal

distribution of Ox immunoreactivity within the total tubular hypothalamus and the LC in the pons (p ≤ 0.037); however, all other pontine nuclei demonstrated a normal distribution (p ≥ 0.10). If a non-normal distribution in the total tuberal hypothalamus and the LC SIDS cases is assumed, based on the Shapiro–Wilk test, post hoc one way-ANOVA of the ‘small’ and ‘large’ reduction groups of SIDS compared to each other and non-SIDS cases, statistically significant differences between all these three groups are observed (p  ≤ 0.047; Fig. 7b). All other pontine nuclei such as the DR and LDT (Fig. 7b) only demonstrated ‘large’ reductions.

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Acta Neuropathol Table 3  Covariate analysis of the SIDS dataset for correlation with decreased OxA immunoreactivity in the tuberal hypothalamus and orexinergic quantifiable fibre staining in the pons Risk factor

DMH

PeF

LH

THT

LC

DR

LDT

DTg

MPB

Pn

Gender Prone Smoke URTI Preterm SIDS 1A Petechiae

0.40 0.33 0.17 0.91 0.26 0.13 0.68

0.48 0.63 0.57 0.83 0.94 0.29 0.17

0.72 0.62 0.29 0.75 0.60 0.98 0.84

0.75 0.97 0.41 0.40 0.80 0.12 0.30

0.36 0.35 0.09 0.10 0.48 0.36 0.30

0.56 0.78 0.61 0.58 0.63 0.94 0.36

0.67 0.50 0.92 0.23 0.38 0.37 0.83

0.35 0.85 0.16 0.69 0.83 0.42 0.49

0.85 0.09 0.59 0.16 0.15 0.34 0.56

0.93 0.96 0.38 0.95 0.77 0.92 0.21

Pulmonary

0.72

0.35

0.65

0.49

0.18

0.55

0.24

0.42

0.34

0.24

DMH dorsal medial hypothalamus, DR dorsal raphe, DTg dorsal tegmental nucleus, LH lateral hypothalamus, LDT laterodorsal tegmental nucleus, LC locus coeruleus, MPB medial parabrachial nucleus, PeF perifornical area, Pn pontine nucleus, THT tuberal hypothalamus

Discussion This study shows Ox immunoreactivity is decreased in the brain of infants who died from SIDS. In the hypothalamus the reduction was up to 21 %, whereas in the pons it was up to 50 %. These decreases did not correlate with a total decrease in neuron numbers or any of the common risk factors for SIDS. Both small (5–10 %) and large (20–30 %) decreases in Ox immunoreactivity in the tuberal hypothalamus were observed. Mechanisms of decreased Ox immunoreactivity There have been several studies examining possible mechanisms that reduce Ox immunoreactivity. Hypoxia and/ or hypercapnia can induce rapid (within a few hours) changes in Ox immunoreactivity. In adult rats exposed to 3 h of hypercapnia (10 % CO2; [91]) or intermittent hypoxia (6–8 % O2) during sleep for a week [55], there was a 50–60 % reduction in PPO mRNA levels in the hypothalamus. A 48-min exposure to intermittent hypercapnic hypoxia (7 % O2/8 % CO2) in 13- to 14-day-old piglets induced a 25 % decrease in Ox immunoreactivity in the hypothalamus with no changes in the total number of neurons in the tuberal hypothalamus [26]. It has also been shown that 6 months’ exposure to ischaemia/reoxygenation injury does not induced changes in active caspase 3 immunoreactivity in Ox neurons or induce histologically identifiable neuronal death; however, ischaemia/reoxygenation injury promoted apoptosis in other (non-Ox) neuronal groups such as the LC and dopaminergic neurons of the midbrain [109]. Ox neurons are not apoptotic under hypoxic [109] or excitotoxicity [78] conditions possibly because of the neuronal protective effect of Ox stimulation [108]. Intracerebroventricular OxA given during ischaemia induced by middle cerebral artery occlusion promoted neuronal

survival [108]; this action is mediated via Ox receptor activation and the PI3K/Akt pathway [92, 93]. Given that (i) Ox neurons are chemosensitive [104], (ii) they self-stimulate via Ox receptor 2 [106] and (iii) stimulation inhibits activation of caspase 3 [93] this neuroprotective pathway appears likely. In addition, active caspase 3 in Ox neurons is correlated with the presence of coarse granular Ox immunoreactive staining [77]. Coarse granular staining was not observed in our Ox neurons. Previous studies by our laboratory have demonstrated that multiple brainstem nuclei demonstrate apoptosis in SIDS [63, 66, 102]. Of these brainstem nuclei, all have been observed to contain Ox neuronal projections [69]. However, Ox neurons project extensively throughout the brain including regions that are not apoptotic in SIDS. It is possible that loss of Ox immunoreactivity may contribute to reduced global neuroprotection in SIDS but not explain why specific brainstem regions are affected. For SIDS cases, the magnitude of the reduction in Ox immunoreactivity varied markedly between the hypothalamus (20 %) compared to the pons (40–60 %). A similar immunoreactive expression pattern (where fibres show a greater loss of immunoreactivity then Ox neurons) has been observed during ageing; Sawai et al. [89] reported that there was a difference between the number of Ox staining neurons (decreased by 10–25 %) and the area of fibre immunoreactivity of this staining (decreased by 60–80 %) in ageing mice. In ageing rhesus monkeys, reduced OxB immunoreactivity was observed in the LC while there was no decline in the hypothalamus [25]. The lack of changes in the hypothalamus in Downs et al. [25] may be due to the age range examined since relatively small (10 %) reduction in Ox immunoreactivity occurs over this age range [45]. A possible explanation for the difference in decreased immunoreactivity could be that SIDS infants have a large release of Ox from axon fibres prior to death, which is not recovered due to inhibition of PPO synthesis by hypoxia or lack

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of vesicle translocation. However, further research into this area is required. While, in our study, there was no correlation between reduced Ox immunoreactivity and the prone sleeping position (prone sleeping result in asphyxia due to airway obstruction [15]), it has been suggested that up to 50 % of SIDS cases may be associated with accidental asphyxiation [97]. This is due to a combination of not only just prone sleeping or/bedsharing [43, 48] but also obstructive events such as obstructive and mixed apnoeas [35] and reflex apnoea following the activation of the laryngeal chemoreflex by aspirated gastric contents [55]. Recently β-amyloid precursor protein was observed to be upregulated in SIDS infants regardless of the mode of death [46], indicating that axonal injury in SIDS infants is not restricted to just prone sleeping infants. Given the similarities (i) decreased Ox immunoreactivity expression and (ii) lack of neuronal loss, in our hypoxia/hypercapnic exposure animals [26], we suggest that ante-mortem hypoxic events may be responsible for changes such as decreased Ox immunoreactivity observed in SIDS infants. SIDS physiology and clinical applications Multiple sleep- and respiration-related abnormalities have been demonstrated in infants who die from SIDS [16–18, 74] and in infants who suffer apparent life-threatening events or “near miss SIDS” [30, 38, 45, 61]. These abnormalities include dysfunctional or immature sleep architecture [16–18, 74], increased apnoeas during REM sleep [33], upper airway obstruction [97], obstructive and mixed apnoeas [36, 42], periodic breathing [50] and impaired arousal thresholds [71]. Dysfunctional sleep Since the reduction in orexinergic neurons we observed (21 %) is similar to that described by Chen et al. [14], who found that a 23 % reduction in Ox immunoreactivity (using siRNA targeting of the PPO gene), results in dysfunctional REM sleep patterns and disrupted sleep architecture, it is possible that the abnormal sleep architecture with altered REM sleep in SIDS infants may be related to a reduction in Ox. Ox neuronal activity consolidates the phase occurrence of REM/non-REM sleep [11, 14], an effect mediated by exogenous release of Ox in the LC, DR and LDT [62]. Loss of Ox signalling decreases the REM to REM sleep interval and increases the duration of REM sleep bouts [12, 13]. Ox signalling in the LC influences the switching between REM and non-REM sleep with impaired Ox signalling resulting in increased bouts of REM sleep with reduced intervals between bouts [13]. Ox receptor 2 signalling in the pontomesenephalic tegmentum (PPT/LDT) inhibits REM sleep

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Acta Neuropathol

[12] and Ox receptor 2 blockade [98] or failure of orexinergic innervation of GABAergic neurons [12] in this region increases REM sleep bout duration. In this study we identified reduced Ox immunoreactivity in these areas. Decreased Ox immunoreactivity was also observed in the DR. Ox excites both serotonin- and GABAergic neurons in the DR and inhibits glutamate excitation of serotonin neurons [41, 60]. Serotonin neurons in the DR are, in part, regulated by surrounding glutamate and GABAergic neurons which modulate serotoninergic innervation of the LDT and PPT to regulate REM/non-REM sleep [73]. It is possible that the reduced innervation of Ox may contribute to impaired regulation of sleep by serotonin neurons. The serotonin neurons in the DR also provide positive feedback to inhibit Ox neurons in the hypothalamus [105]. In SIDS there is a reduction of serotonin content in the hypothalamus [94] which could be indicative of reduced feedback from the DR (one of the main site of serotonin production) which has been found to have altered serotonergic expression in SIDS [27, 53, 64]. Finally, we also noted reduced orexinergic fibres in the MPB, part of the parabrachial complex, which also contributes to REM/non-REM sleep [70]. There is a gradual decrease in the amount of sleep time occupied by REM sleep in healthy infants from 50 % at 1 month of age to 30 % at 6 months of age [39]. In SIDS infants, this decline is more gradual [39] and, although there is no increase in total REM sleep in infants who survive life-threatening events [34, 86], an increase in REM sleep has been observed in 0- to 2-month-old infants that later succumbed to SIDS [18, 91] specifically between 3 a.m. and 7 a.m. [85]. Schechtman et al. [91] suggested that the increased time spent in REM sleep (along with increased quiet sleep and decreased awakenings) in their 0- to 2-month-old infants that later succumbed to SIDS was due to “an exaggeration of the normal circadian pattern” or reduced awakenings between 3 and 7 a.m. In agreement with Schechtman et al. [91], Cornwell et al. [16–18] observed increased REM sleep in infants at risk of SIDS between 2 and 5 a.m. Further studies should be performed to examine this discrepancy. Another study showed increased latency to REM sleep and impaired non-REM sleep in infants which later succumb to SIDS [17, 72], suggesting a change in sleep architecture rather than inhibition of different sleep states. Finally, the tonic inhibitory phasic output from the PPT/LDT that induces REM/nonREM sleep transition was decreased in a single SIDS infant examined by Kohyama et al. [57]. Previous studies have demonstrated that other neurochemicals involved in REM sleep regulation are altered in SIDS. Melatonin in the CSF is decreased in SIDS infants [95]. Reduced serotonin activity occurs in SIDS [27, 53, 79], and a recent animal study [20] demonstrated that

Acta Neuropathol

inhibiting serotonergic neuronal activity in the paragigantocellularis of the medulla results in dysfunctional sleep by reducing REM sleep. As discussed previously there have been contrasting findings regarding changes in REM/nonREM sleep in SIDS; however, all these studies have shown that REM/non-REM sleep is abnormal compared to controls, these neurochemical factors may contribute to this. Respiration during sleep and arousal responses Ox is involved in the development and function of sleep cycle regulation [3] and respiratory control during sleep [30]. There is no evidence that the decrease in Ox immunoreactivity seen in this study (20 %) may induce impairment of respiration (sleep apnoea) during sleep under basal or asphyxia conditions [23, 24, 75]. Increased incidence of sleep apnoeas have only been seen with complete 100 % loss of Ox immunoreactivity [75]. It is still possible given the multitude of abnormal neurotransmitters and receptors in SIDS [65] that Ox may contribute to respiratory compromise in SIDS. Research should be directed towards determining what level of decreased Ox immunoreactivity induces central sleep apnoea. Central sleep apnoea may occur in Ox, or BDNF knockout mice [2, 75], and is also promoted by serotonin receptor antagonists [107]; the frequency of apnoeas was similar in all these neurochemical/ factor impairments suggesting that they may impair a common neuronal system. Multiple respiratory nuclei receive innervation from both Ox and serotonin as well as express BDNF [68, 100, 103]. For example, in the pre-Bötzinger complex impairment of the neurokinin 1 receptor neurons, as well as impaired Ox and serotonin signalling, results in central sleep apnea. Impaired blockage of Ox and serotonin receptors in the Bötzinger complex would confirm this. In SIDS there have been descriptions of impaired serotonin and BDNF expression (and now Ox) and apoptosis of preBötzinger nuclei [58, 65]. The orexinergic system forms part of the ascending arousal system, and contributes to arousal under both basal and stressful conditions [56, 87]. Intracerebroventricular administration of OxA induces arousal from sleep [40], while narcoleptic patients demonstrate impaired arousal thresholds [6]. However, the increased sleep drive in narcolepsy could contribute to this effect [90]. Ox ablation mice demonstrate delayed emergence and arousal from anaesthetics compared to wild-type mice [51]. Under basal conditions this involves orexinergic input to the LC, DR, tuberal mammillary and LDT/PPT nuclei as well as the cholinergic neurons of the basal forebrain [1]. During asphyxia, orexinergic innervation also involves the retrotrapezoid nucleus, C1 neurons, parabrachial/KöllikerFuse subnuclei, nucleus of the solitary tract and the Bötzinger complex; the key neuronal groups for arousal under

asphyxia are proposed to be the LC, DR, retrotrapezoid nucleus and C1 neurons [38]. Orexinergic neurons provide direct excitatory input to these nuclei, to reduce the threshold for neuronal excitation and sustain this excitation [56, 59]. It has been suggested that Ox may have a minimal role in inducing arousal under asphyxia conditions given other neuronal systems involved [38], also supported by more recent research, [9, 10] showing that optogenetic modulation/excitation of the LC during sleep in rats promoted immediate arousal while the same stimulation of Ox neurons only induced arousal after a 30-s delay, suggesting that the LC (presumably via noradrenergic innervation) provides faster and greater contribution to arousal then Ox neurons. In the context of SIDS, two points are important. First multiple neurochemical abnormalities are known to occur in SIDS [65], many of which are part of the arousal signalling pathways described by Guyenet and Abbott [38]. It has been suggested that SIDS infants do not demonstrate an arousal response prior to the onset of gasping [55] indicating impairment of arousal. Although Ox plays a minor role in arousal from asphyxia, it is still part of the complex of neuronal groups contributing to arousal. Second, studies of infants who subsequently died from SIDS demonstrate impaired cortical arousals (compared to sub-cortical arousals) [48]. Ox neurons have recently been shown to be innervated by C1 neurons and act as one of the mediators to promote cortical arousal [4]. This research group has also shown that C1 neurons are activated by acute hypoxia [37] with in vivo excitation demonstrating very similar physiological responses to acute hypoxia such as cardiorespiratory stimulation, sighs and arousal [8].

Conclusion We provide, for the first time, evidence of a role of the orexinergic system in SIDS, whereby the immunoreactivity of Ox is decreased by up to 21 % in the hypothalamus with a corresponding loss of up to 50 % of orexin immunoreactive fibres in the pons. These findings did not correlate with any individual common risk factor for SIDS. However, questions remain about how immunoreactivity is lost. Given the evidence at hand we suggest this is mediated by hypoxic insults. The differences in the amount of Ox immunoreactivity lost within this study suggest that multiple factors may influence this reduced immunoreactivity. In view of the important role of Ox in sleep regulation, our demonstration of decreased Ox immunoreactivity in sleep-related pontine nuclei in infants who die from SIDS suggests that the abnormalities in sleep regulation demonstrated in infants who subsequently die from SIDS, may, in part, be due to changes in Ox.

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Acknowledgments  The tissue used in this study was provided by the NSW Forensic and Analytical Science Service. The authors acknowledge the facilities, and scientific and technical assistance of the Australian Microscopy and Microanalysis Research Faculty at the Australian Centre of Microscopy and Micro Analysis, University of Sydney. Research funded by the SIDS Stampede, Australia, and the Miranda Bradshaw Foundation. Conflict of interest  The authors report no conflicts of interest.

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