Quantifying GFAP immunohistochemistry in the brain - Introduction of the Reactivity score (R-score) and how it compares to other methodologies.

BACKGROUND: Immunohistochemical upregulation of glial fibrillary acidic protein (GFAP) is commonly used to detect astrogliosis in tissue sections and includes measurement of intensity and/or distribution of staining. There remains a lack of standard objective measures when diagnosing astrogliosis and its severity. NEW METHOD: Aim was to test a novel semi-quantitative assessment of GFAP which we term reactivity (R)-score, on its reproducibility and sensitivity to measure astrogliosis. The R-score, which is based on the proportion of astrocytes seen at each level of reactivity, was compared to 3 other commonly employed quantification methods in research: (1) thresholding, (2) point-counting, and (3) qualitative grading. Sub-regions of the hippocampus, medulla, and cerebellum were studied in piglet, and 4 human cases with clinically reported astrogliosis. Intra-assay coefficient of variation (CV) and percentage agreement cut-offs of ≤ 20% and ≥ 75% were used respectively to compare amongst the methods, with outcome measures being reproducibility across serial and non-serial sections, resilience to changes in experimental conditions, and inter- and intra-rater concordance. RESULTS: Averaged across 3 brain regions, the intra-assay coefficient of variation (CV) was 5% for R-score, with inter and intra-rater kappa scores being 0.99 and 0.95 respectively. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: Based on CV values, the R-score was superior to thresholding (CV of 51%) and point-counting (CV of 16%), with the qualitative grade being found to be on par (percentage agreement 95%). Given the ease, reproducibility and selectivity of the R-score, we propose its validity in future research purposes and clinical application.

Choline-acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the human infant dorsal motor nucleus of the Vagus (DMNV), and alterations according to sudden infant death syndrome (SIDS) category.

Amongst other molecules, the cholinergic system consists of choline-acetyltransferase (ChAT, - synthesis enzyme), acetylcholinesterase (AChE - primary hydrolysis enzyme), and butyrylcholinesterase (BuChE - secondary hydrolysis enzyme). In the brainstem, the Dorsal Motor Nucleus of The Vagus (DMNV) has high cholinergic expression and is a region of interest in the neuropathology of sudden infant death syndrome (SIDS). SIDS is the unexpected death of a seemingly healthy infant, but postmortem brainstem abnormalities suggesting altered cholinergic regulation have been found. This study aimed to determine the percentage of positive ChAT and AChE neurons within the infant DMNV through immunohistochemistry at the three levels of the brainstem medulla (caudal, intermediate, and rostral), to investigate whether the proportion of neurons positive for these enzymes differs amongst the diagnostic subgroups of SIDS compared to those with an explained cause of Sudden unexpected death in infancy (eSUDI), and whether there were any associations with SIDS risk factors (male gender, cigarette smoke exposure, co-sleeping/bed sharing, and prone sleeping). Results showed that ChAT-positive neurons were lower in the rostral DMNV in the SIDS II cohort, and within the caudal and intermediate DMNV of infants who were exposed to cigarette smoke. These findings suggest altered cholinergic regulation in the brainstem of SIDS infants, with potential contribution of cigarette smoke exposure, presumably via the nicotinic acetylcholinergic receptor system.

Effects of e-vapour and high-fat diet on the immunohistochemical staining of nicotinic acetylcholine receptors, apoptosis, microglia and astrocytes in the adult male mouse hippocampus.

The use of e-cigarettes/e-vapour, and the consumption of a high-fat diet (HFD), are two popular lifestyle choices associated with alterations in the hippocampus. This study, using a mouse model, investigated the effects of exposure to e-vapour (± nicotine) and HFD (43% fat) consumption, on the expression of nicotinic acetylcholine receptor (nAChR) subunits α3, α4, α7 and ß2, apoptosis markers caspase-3 and TUNEL, microglial marker Iba-1, and astrocyte marker GFAP, in hippocampal subregions of dentate gyrus (DG) and cornu ammonis (CA) 1-3. The major findings included: (1) HFD alone had minimal effect with no consistent pattern or interaction between the markers, (2) E-vapour (± nicotine) predominantly affected the CA2 subregion, decreasing α7 and ß2 nAChR subunits and Iba-1, (3) Nicotine e-vapour increased TUNEL across all subregions, and (4) HFD, in the presence of nicotine-free e-vapour, decreased caspase-3 and increased TUNEL across all regions, and decreased Iba-1 in the CA subregions, while HFD and nicotine-containing e-vapour, subregion specifically affected the α3, α4 and α7 nAChR subunits, with a protective effect against change in GFAP in the DG and Iba-1 in the CA1 and CA3. These findings highlight that e-vapour itself alters nAChRs, particularly in the CA2 subregion, associated with a decrease in neuroinflammatory response (Iba-1) across the whole hippocampus, and the addition of nicotine increases cell apoptosis across the whole hippocampus. HFD alone was not detrimental in our model, but in the presence of nicotine-free e-vapour, it differentially affected apoptosis, while the addition of nicotine increased nAChR subunits.

Cell death in the lateral geniculate nucleus, and its possible relationship with nicotinic receptors and sudden infant death syndrome (SIDS).

The role of the lateral geniculate nucleus (LGN) in vision has been extensively studied, yet its extraretinal capacities are still being investigated, including its role in arousal from sleep. The ß2 nicotinic acetylcholine receptor (nAChR) subunit is involved in the laminal organisation of the LGN with magnocellular (MC) and parvocellular (PC) neurons. Sudden infant death syndrome (SIDS) occurs during a sleep period and, neuropathologically, is associated with increased neuronal cell death and altered nAChRs. A recent qualitative pilot study from our group implicates the possibility of increased neuronal death/apoptosis in the SIDS LGN. The present study used quantitative analysis to report the baseline expression of apoptotic and nAChR subunits α7 and ß2 in the PC and MC layers of the LGN, to determine correlations amongst these markers within layers and across layers, and to evaluate changes in the expression of these markers in the LGN of SIDS infants, along with associations with SIDS risk factors, such as age, sex, cigarette smoke exposure, bed-sharing, and presence of an upper respiratory tract infection (URTI). Tissue was immunohistochemically stained for cell death markers of active caspase-3 (Casp-3) and TUNEL, and for the α7 and ß2 nAChR subunits. Amongst 43 cases of sudden and unexpected deaths in infancy (SUDI), classifications included explained deaths (eSUDI, n = 9), SIDS I (n = 5) and SIDS II (n = 29). Results indicated a strong correlation of the apoptotic markers and ß2 nAChR subunit between the LGN layers, but not across the markers within the layers. Amongst the diagnostic groups, compared to eSUDI, the SIDS II cases had decreased Casp-3 expression while ß2 nAChR expression was increased in both PC and MC layers. Amongst the SIDS risk factors, URTI and bed-sharing were associated with changes in neuronal death but not in the α7 and ß2 markers. In conclusion, our findings do not support a role for the α7 and ß2 nAChRs in apoptotic regulation of the LGN layers during infancy. However, for SIDS victims, an inverse correlation between the changes for markers of apoptosis and the ß2 nAChR subunit expression suggests altered LGN function.

Immunohistochemistry for acetylcholinesterase and butyrylcholinesterase in the dorsal motor nucleus of the vagus (DMNV) of formalin-fixed, paraffin-embedded tissue: comparison with reported literature.

The majority of research regarding the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain has been conducted using histochemistry to identify enzymatic activity in frozen fixed tissue. However, retrospective human neurochemistry studies are generally restricted to formalin-fixed, paraffin-embedded (FFPE) tissues that are not suitable for histochemical procedures. The availability of commercially available antibody formulations provides the means to study such tissues by immunohistochemistry (IHC). In this study, we optimised IHC conditions for evaluating the expression of AChE and BuChE in the brainstem, focusing on the dorsal motor nucleus of the vagus, in human and piglet FFPE tissues, using commercially available antibodies. Our results were compared to published reports of histochemically determined AChE and BuChE expression. We varied antibody concentrations and antigen retrieval methods, and evaluated different detection systems, with the overall aim to optimise immunohistochemical staining. The primary findings, consistent across both species, are: (1) AChE and BuChE expression dominated in the neuronal somata, specifically in the neuronal cytoplasm; and (2) no change in the protocol resulted in axonal/neuropil expression of AChE. These results indicate that IHC is a suitable tool to detect AChE and BuChE in FFPE tissue using commercial antibodies, albeit the staining patterns obtained differed from those using histochemistry in frozen tissue. The underlying cause(s) for these differences are discussed in detail and may be associated with the principal components of the staining method, the antibody protein target and/or limitations to the detection of epitopes by tissue fixation.

Three-dimensional orthodontic imaging in children across the age spectrum and correlations with obstructive sleep apnea.

STUDY OBJECTIVES: To determine baseline facial convexity measurements in children with obstructive sleep apnea (OSA) across the age spectrum. METHODS: Polysomnogram, stereophotogrammetry, and biometric data were collected from children aged 0-18 years who were being investigated for OSA. Analyses evaluated differences in facial convexity according to OSA severity and other sleep parameters, while adjusting for age, ethnicity, and sex. RESULTS: Ninety-one children, aged 0.05-16.02 years, met the inclusion criteria for this study. Initial analysis showed that the logarithm of age had a significant effect on facial convexity (P = 8.3·10-7) with significant effects of sex (P = 1.3·10-2), while excluding OSA. Ordinal logistic regression taking into consideration age, sex, weight, height, and ethnicity with OSA severity categorized as obstructive apnea-hypopnea index negative, mild, moderate, or severe showed that facial convexity was associated with OSA severity (P = 2.2·10-3); an increasing obtuse angle of convexity increased the tendency to be classified as having severe OSA. CONCLUSIONS: Using three-dimensional imaging, we found an added impact of infancy on changes of facial convexity with age. While modeling could describe facial convexity without any OSA-associated sleep parameters, differences in facial convexity were present among groups with different levels of OSA severity adjusted for growth (age, weight, and height), sex, and ethnicity. The method provides a safer and cheaper alternative to other medical imaging techniques in children and holds potential for future use in studies of craniofacial structure. CITATION: Tyler G, Machaalani R, Waters KA. Three-dimensional orthodontic imaging in children across the age spectrum and correlations with obstructive sleep apnea. J Clin Sleep Med. 2023;19(2):275-282.

Early Postnatal Exposure to Intermittent Hypercapnic Hypoxia (IHH), but Not Nicotine, Decreases Reelin in the Young Piglet Hippocampus.

This study evaluated the expression of reelin, an extracellular protein involved in lamination and migration of neurons, in the hippocampus of young piglets, and quantified to examine the following: (i) baseline levels within layers of the hippocampus and dentate gyrus (DG); (ii) differences between ventral and dorsal hippocampi; and (iii) changes attributable to postnatal exposure to continuous nicotine for 12 days, or intermittent hypercapnic hypoxia (IHH), with further analysis according to duration of IHH (1 vs 4 days). Additionally, we analysed whether any exposure altered DG morphology and whether it is related to altered reelin expression. Reelin was visualised via immunohistochemistry, and the number of positive reelin cells/mm2 was measured in the CA4/Hilus, layers of the DG, and the CA1. The dorsal DG had significantly more reelin within the subgranular zone compared to the ventral DG (p < 0.01). There was no difference in reelin between nicotine (n = 5) and controls (n = 5). IHH exposed piglets (n = 10) had significantly lowered reelin in the CA1 (p = 0.05), specifically the stratum pyramidale (p = 0.04) and the hippocampal fissure (p = 0.02), compared to their controls (n = 7); the duration of IHH had no effect. No exposure was associated with an alteration in DG morphology. This study shows that postnatal IHH exposure decreased reelin expression in the developing piglet hippocampal CA1, suggesting that IHH may result in altered neuronal migration.

Impact of High Fat Consumption on Neurological Functions after Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) and obesity are two common conditions in modern society; both can impair neuronal integrity and neurological function. However, it is unclear whether the coexistence of both conditions will worsen outcomes. Therefore, in a rat model, we aimed to investigate whether the coexistence of TBI and a high-fat diet (HFD) has an additive effect, leading to more severe neurological impairments, and whether they are related to changes in brain protein markers of oxidative stress, inflammation, and synaptic plasticity. Sprague-Dawley rats (female, ∼250 g) were divided into HFD (43% fat) and diet (CD) (17% fat) groups for 6 weeks. Within each dietary group, half underwent a TBI by a weight-drop device, and the other half underwent sham surgery. Short-term memory and sensory function were measured at 24 h, 1 week, 3 weeks, and 6 weeks post-TBI. Brain tissues were harvested at 24 h and 6 weeks post-TBI, and markers of oxidative stress, apoptosis, inflammation, and synaptic plasticity were measured via immunostaining and Western blotting. In rats without TBI, HFD increased the pre-synaptic protein synaptophysin. In rats with TBI, HFD resulted in worsened sensory and memory function, an increase in activated macrophages, and a decrease in the endogenous antioxidant manganese superoxide dismutase (MnSOD). Our findings suggest that the additive effect of HFD and TBI worsens short term memory and sensation deficits, and may be driven by enhanced oxidative stress and inflammation.

Genes involved in paediatric apnoea and death based on knockout animal models: Implications for sudden infant death syndrome (SIDS).

The mechanism of death in Sudden infant death syndrome (SIDS) remains unknown but it is hypothesised that cardiorespiratory failure of brainstem origin results in early post-natal death. For a subset of SIDS infants, an underlying genetic cause may be present, and genetic abnormalities affecting brainstem respiratory control may result in abnormalities that are detectable before death. Genetic knockout mice models were developed in the 1990s and have since helped to elucidate the physiological roles of a number of genes. This systematic review aimed to identify which genes, when knocked out, result in the phenotypes of abnormal cardiorespiratory control and/or early post-natal death. Three major genes were identified: Pet1- a serotonin transcription factor, the neurotrophin pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor (PAC1). Knockouts targeting these genes had blunted hypercapnic and/or hypoxic responses and early post-natal death. The hypothesis that these genes have a role in SIDS is supported by their being identified as abnormal in SIDS cohorts. Future research in SIDS cohorts will be important to determine whether these genetic abnormalities coexist and their potential applicability as biomarkers.

Morphology of the Dentate Gyrus in a Large Cohort of Sudden Infant Deaths-Interrelation Between Features but Not Diagnosis.

Morphological differences in the dentate gyrus (DG) have been reported in sudden unexpected deaths in infancy (SUDI), with the feature of focal granule cell (GC) bilamination (FGCB) reported as increased in unexplained SUDI, including sudden infant death syndrome (SIDS), compared with explained SUDI (eSUDI). However, it remains to be determined how these morphologies relate to each other and their extent along the anteroposterior length. This retrospective study evaluated the prevalence of FGCB, single or clustered ectopic GCs, granule cell dispersion (GCD), heterotopia, hyperconvolution, gaps, thinning, blood vessel dissection (BVD), and cuffing (BV cuffing), in an Australian SUDI cohort, and compared the prevalence of these features in eSUDI and unexplained SUDI. We analyzed 850 formalin-fixed paraffin-embedded serial and subserial sections of the hippocampus at the level of the lateral geniculate nucleus from 90 infants, and identified GCD in 97% of infants, single ectopic cells, hyperconvolution, thinning, and BVD in 60%-80%, heterotopia in 36%, gaps, clusters of ectopic cells and BV cuffing in 9%-15%, and FGCB in 18%. These features are clustered within 3-5 serial sections. The presence of FGCB correlated with single ectopic GCs and hyperconvolution. There were no differences in the prevalence of these features between unexplained SUDI (n = 74) and eSUDI (n = 16). Our findings highlight that DG morphological features are highly localized, extending 14-35 µm at their focal location(s) along the anteroposterior length. Consequently, multiple sections along the longitudinal extent are required to identify them. No feature differentiated SUDI from eSUDI in our cohort, thus we cannot conclude that any of these features are abnormal and it remains to be determined their functional significance.

Relationship between sleep respiration, architecture and childhood enuresis: Correlates between polysomnography and questionnaire.

AIM: Nocturnal enuresis (NE) and sleep-disordered breathing (SDB) are common in childhood. While the two disorders are linked, those links are still being clarified. METHODS: This study compared sleep profiles and enuresis-related behaviours between children with NE and those without, who were referred to a tertiary sleep unit with suspected SDB, using the combination of polysomnography (PSG) and questionnaire. Continuous numerical data were analysed after adjusting for body mass index z-score. RESULTS: The study included 52 Children (39 boys, 13 girls) aged 5-14 years. Twenty-one had enuresis (10 monosymptomatic enuresis (MNE) and 11 non-monosymptomatic enuresis (NMNE)) and 31 did not have enuresis. The majority had comorbidities. On PSG, all children with NE had moderate obstructive sleep apnoea (OSA) compared to the control group which were of mild OSA. Children with NMNE had a higher percentage time in stage-3 non-REM sleep when compared to the non-enuretic and MNE groups (P < 0.05). On the questionnaire, more parents of the NE groups reported that their child was 'difficult to wake in the morning' (P < 0.05). CONCLUSION: In this heterogeneous population referred for suspected SDB, children with NE had moderate OSA, yet those with MNE had increased arousals and more often report difficulty waking than children with suspected SDB who do not wet, while children with NMNE exhibit changes in sleep architecture suggesting deeper sleep. These differences may impact treatment choices for children with enuresis.

Immunostaining for NeuN Does Not Show all Mature and Healthy Neurons in the Human and Pig Brain: Focus on the Hippocampus.

Neuronal nuclei (NeuN) is a neuron-specific nuclear protein, reported to be stably expressed in most postmitotic neurons of the vertebrate nervous system. Reduced staining has been interpreted by some to indicate loss of cell viability in human studies, while others suggest this may be because of changes in the antigenicity of the target epitope. Preliminary studies in our laboratory found low immunostaining for the NeuN antibody on formalin fixed and paraffin embedded (FFPE) human brain tissue. We report on the techniques and results used to enhance the staining for NeuN in that tissue. In parallel, we stained NeuN in piglet brain tissue, sourced from an experimental model where methodological parameters, including those for tissue fixation and storage, were tightly controlled. In human FFPE brain tissue, we were unable to enhance NeuN immunostaining to a degree sufficient for cell counting. In contrast, we found consistently high levels of staining in the piglet brain tissue. We conclude that processes used for fixation and storage of human FFPE brain tissue are responsible for the reduced staining. These results emphasize that a cautionary approach should be taken when interpreting NeuN staining outcomes in human FFPE brain tissue.

Expression of reelin with age in the human hippocampal formation.

Reelin plays a key role in neuronal migration and lamination in the cortex and hippocampus. Animal studies have shown that reelin expression decreases with age. The aim of this study was to evaluate the expression of reelin in all layers of the human hippocampal formation across three age groups. We used immunohistochemistry in formalin fixed and paraffin embedded hippocampal tissue from infants (1-10 months; n = 9), children (4-10 years; n = 4), and adults (45-60 years; n = 6) to stain for reelin. Expression was quantified (measured as the number of positive reelin cells/mm2 ) in the granule cell layer of the dentate gyrus (DG), the molecular layer of the dentate gyrus (ML), the hippocampal fissure (HF), stratum lacunosum moleculare (SLM), CA4/Hilus and the stratum pyramidale layer of CA3, CA2, and CA1. Expression of reelin was highest in the HF irrespective of age, followed by the SLM and ML. Minimal to no expression was seen in the stratum pyramidale layer of CA1-3. With age, reelin expression decreased and was statistically significant from infancy to childhood in the HF (p = .02). This study confirms that reelin expression decreases with age in the human hippocampus, and shows for the first time that the major decrease occurs between infancy and early childhood.

The Unfolded Protein Response in the Human Infant Brain and Dysregulation Seen in Sudden Infant Death Syndrome (SIDS).

Low orexin levels in the hypothalamus, and abnormal brainstem expression levels of many neurotransmitter and receptor systems in infants who died suddenly during a sleep period and diagnosed as sudden infant death syndrome (SIDS), may be linked to abnormal protein unfolding. We studied neuronal expression of the three unfolded protein response (UPR) pathways in the human infant brainstem, hypothalamus, and cerebellum: activating transcription factor 6 (ATF6), phosphorylated inositol-requiring enzyme 1 (IRE1), and phosphorylated protein-kinase (PKR)-like endoplasmic reticulum (ER) kinase (pPERK). Percentages of positively stained neurons were examined via immunohistochemistry and compared between SIDS (n = 28) and non-SIDS (n = 12) infant deaths. Further analysis determined the effects of the SIDS risk factors including cigarette smoke exposure, bed-sharing, prone sleeping, and an upper respiratory tract infection (URTI). Compared to non-SIDS, SIDS infants had higher ATF6 in the inferior olivary and hypoglossal nuclei of the medulla, higher pIRE1 in the dentate nucleus of the cerebellum, and higher pPERK in the cuneate nucleus and hypothalamus. Infants who were found prone had higher ATF6 in the hypoglossal and the locus coeruleus of the pons. Infants exposed to cigarette smoke had higher ATF6 in the vestibular and cuneate nuclei of the medulla. Infants who were bed-sharing had higher pPERK in the dorsal raphe nuclei of the pons and the Purkinje cells of the cerebellum. This study indicates that subgroups of SIDS infants, defined by risk exposure, had activation of the UPR in several nuclei relating to proprioception and motor control, suggesting that the UPR underlies the neuroreceptor system changes responsible for these physiological functions, leading to compromise in the pathogenesis of SIDS.

Brain health is independently impaired by E-vaping and high-fat diet.

Tobacco smoking and high-fat diet (HFD) independently impair short-term memory. E-cigarettes produce e-vapour containing flavourings and nicotine. Here, we investigated whether e-vapour inhalation interacts with HFD to affect short-term memory and neural integrity. Balb/c mice (7 weeks, male) were fed a HFD (43% fat, 20 kJ/g) for 16 weeks. In the last 6 weeks, half of the mice were exposed to tobacco-flavoured e-vapour from nicotine-containing (18 mg/L) or nicotine-free (0 mg/L) e-fluids twice daily. Short-term memory function was measured in week 15. HFD alone did not impair memory function, but increased brain phosphorylated (p)-Tau and astrogliosis marker, while neuron and microglia levels were decreased. E-vapour exposure significantly impaired short-term memory function independent of diet and nicotine. Nicotine free e-vapour induced greater changes compared to the nicotine e-vapour and included, increased systemic cytokines, increased brain p-Tau and decreased postsynaptic density protein (PSD)-95 levels in chow-fed mice, and decreased astrogliosis marker, increased microglia and increased glycogen synthase kinase levels in HFD-fed mice. Increased hippocampal apoptosis was also differentially observed in chow and HFD mice. In conclusion, E-vapour exposure impaired short-term memory independent of diet and nicotine, and was correlated to increased systemic inflammation, reduced PSD-95 level and increased astrogliosis in chow-fed mice, but decreased gliosis and increased microglia in HFD-fed mice, indicating the inflammatory nature of e-vapour leading to short term memory impairment.

Microglia in the human infant brain and factors that affect expression.

The present study reports on the microglial populations present in 34 regions of the human infant brain (1-11 months), and whether developmental parameters or extrinsic factors such as cigarette smoke exposure, prone sleeping and an upper respiratory tract infection (URTI) influence their expression. Further, we compare microglia populations amongst three sudden unexpected death in infancy (SUDI) sub-groups: explained SUDI (eSUDI, n â€‹= â€‹7), sudden infant death syndrome (SIDS) I (n â€‹= â€‹8) and SIDS II (n â€‹= â€‹13). Ionised calcium binding adaptor molecule-1 (Iba1) was used to determine the morphology and area covered by microglia in a given brain region. Activation was explored using cluster-of-differentiation factor 68 (CD68) and human leukocyte antigen-DP,DQ,DR (HLA). We found regional heterogeneity in the area covered and activation status of microglia across the infant brain. The hippocampus, basal ganglia, white matter and dentate nucleus of the cerebellum showed larger areas of Iba1, while the brainstem had the smallest. Microglia in regions of the basal ganglia and cortex demonstrated positive correlations with infant developmental parameters, while in nuclei of the rostral medulla, negative correlations between microglia parameters were seen. URTI and cigarette smoke exposure were associated with a reduced microglial area in regions of the hippocampus and cortex (parietal and occipital), respectively. In the context of SIDS, a reduced microglial area was seen in SIDS II and fewer SIDS I infants demonstrated activated phenotypes in the hippocampus. Overall, we identify the distribution of microglia in the infant brain to be heterogenous, and influenced by intrinsic and extrinsic factors, and that the SIDS I group is a useful control group for future research into other infant CNS pathologies.

Effects of prenatal cigarette smoke exposure on BDNF, PACAP, microglia and gliosis expression in the young male mouse brainstem.

Cigarette smoke exposure during pregnancy into infancy affects brain growth and development in both short and long term (into adulthood). Using a mouse model of pre- into post- natal cigarette smoke exposure (SE), we aimed to determine the effects on brain derived neurotrophic factor (BDNF) and its receptor TrkB, neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor PAC1, and astrocyte (GFAP) and microglia (Iba-1) immunohistochemical expression, in seven nuclei of the medulla and the facial (FAC) nucleus of the pons. Male pups of dams exposed to two cigarettes (nicotine <1.2 mg, CO <15 mg) twice daily for six weeks prior to mating, during gestation and lactation (n = 5; SE), were compared to pups exposed to air under the same condition (n = 5; SHAM) at postnatal day 20. Expression changes were only evident for BDNF, TrkB and PAC1 and included decreased BDNF in the hypoglossal (XII) nucleus and nucleus of the solitary tract (NTS), increased TrkB in XII but decreased TrkB in the FAC, and increased PAC1 in 4 nuclei of the medulla including the NTS. These results suggest that the effect of SE on the brainstem are region and marker selective, affecting regions of respiratory control (XII and NTS), and restricted to the BDNF system and PAC1, with no effect on activation states of astrocytes or microglia.

The α3 and α4 nicotinic acetylcholine receptor (nAChR) subunits in the brainstem medulla of sudden infant death syndrome (SIDS).

SIDS occurs in early infancy and predominantly during a sleep period. Abnormalities in nicotine receptor binding and in the expression of the nicotinic acetylcholine receptor (nAChR) subunits α7 and ß2 have been reported in the brainstem of SIDS infants. This study focuses on the α3 and α4 nAChR subunits as α3 is important for early postnatal survival while α4 is crucial for nicotine-elicited antinociception and sleep-wake cycle regulation. Tissue from the rostral medulla of infants who died with a known cause of death (eSUDI, n = 7), and from SIDS classified as SIDS I (n = 8) and SIDS II (n = 27), was immunohistochemically stained for the α3 and α4 nAChR subunits and quantified in 9 nuclei comparing amongst these groups. The association with risk factors of sex, cigarette smoke exposure, upper respiratory tract infection (URTI), prone sleeping and bedsharing was also evaluated. Results showed that only α4 changes (increase) were evident in SIDS, occurring in the hypoglossal and cuneate nuclei of SIDS II infants and the nucleus of the spinal trigeminal tract of SIDS I infants. Amongst the SIDS infants, cigarette smoke exposure was only associated with decreased α4 in cribriform fibre tracts, while sex and bedsharing were associated with increases in α3 in the dorsal motor nucleus of the vagus and solitary nucleus, respectively. Combined, these findings suggest that abnormalities in endogenous acetylcholine synthesis and regulation may underlie the altered α3 and α4 nAChR subunit expressions in the SIDS brainstem medulla since the changes were not related to cigarette smoke exposure.

Cell death in the human infant central nervous system and in sudden infant death syndrome (SIDS).

The brainstem has been a focus of sudden infant death syndrome (SIDS) research with amassing evidence of increased neuronal apoptosis. The present study extends the scope of brain regions examined and determines associations with known SIDS risk factors. Immunohistochemical expression of cell death markers, active caspase-3 and TUNEL, was studied in 37 defined brain regions in infants (aged 1-12 months) who died suddenly and unexpectedly (SUDI). A semi-quantitative mean score of marker expression was derived for each region and scores compared between three SUDI subgroups: explained SUDI (eSUDI; n = 7), SIDS I (n = 8) and SIDS II (n = 13). In eSUDI, active caspase-3 scores were highest in several nuclei of the rostral medulla, and lowest in the hypothalamus and cerebellar grey matter (GM). TUNEL was highest in regions of the hippocampus and basal ganglia, and lowest in the thalamus and cerebellar GM. TUNEL scores were higher in SIDS II compared to eSUDI in the amygdala (p = 0.03) and 5/9 nuclei in the rostral medulla (p = 0.04 - 0.01), and higher in SIDS II compared to SIDS I in the amygdala (p < 0.01), putamen (p = 0.01), lentiform nucleus (p = 0.03) and parietal (p = 0.03) and posterior frontal (p = 0.02) cortex. Active caspase-3 was greater in the hypoglossal nucleus (p = 0.03) of SIDS I compared to eSUDI infants. Co-sleeping, cigarette smoke exposure and the presence of an upper respiratory tract infection in SIDS infants was associated with differences in marker expression. This study affirms the sensitivity of the brainstem medulla to cell death in SIDS, and highlights the amygdala as a new region of interest.