Cholinergic Immunohistochemistry in the Brainstem, Focusing on the Dorsal Motor Nucleus of the Vagus (DMNV), and changes within Sudden Infant Death Syndrome (SIDS)

Abstract

Introduction: A dominating hypothesis of Sudden Infant Death Syndrome (SIDS), which is defined as the unexpected death, usually during sleep, in seemingly healthy infants under the age of 1 year, posits that it is due to an abnormal cardiorespiratory regulation via autonomic nuclei within the brainstem medulla. SIDS falls under the category of Sudden Unexpected Deaths in Infancy (SUDI) and has two classifications: SIDS I being an infant with a normal clinical history and their death is not associated with SIDS risk factors, and SIDS II being an infant whose death is associated with risk factors such as unsafe sleeping environment. The Dorsal Motor Nucleus of the Vagus (DMNV) within the brainstem medulla is the main source of parasympathetic vagal output via the vagus nerve and is responsible for cardiorespiratory regulation, which it does through neurotransmitters such as acetylcholine (ACh). ACh levels are identified through the expression of its synthesis enzyme Choline Acetyltransferase (ChAT), and the primary and secondary hydrolysis enzymes Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE). Decreased ChAT in SIDS DMNV has been reported, but no subsequent study has verified this. Moreover, AChE or BuChE expression in the DMNV, to provide a holistic analysis of the ACh system simultaneously, has not been studied. Finally, SIDS is associated with risk factors such as sex, Cigarette Smoke Exposure (CSE), Upper Respiratory Tract Infection (URTI), bed sharing, and sleeping prone, yet their role on ACh marker expression, also remains to be determined. Aims: There were two aims of this study. First was to determine specificity of AChE and BuChE expression in our formalin-fixed and paraffin embedded (FFPE) brain tissue specifically from the medulla, cerebellum, and pons of the infant via immunohistochemistry (IHC), where SIDS and non-SIDS (control case) tissue was used. Traditionally, AChE and BuChE studies are restricted to histochemistry on frozen fixed tissue, but this method is not applicable on FFPE tissue which is how our tissue is stored and available for usage. Second, to determining whether expression of ChAT, AChE, and BuChE within the infant DMNV is altered within SIDS infants and/or by the presence of the risk factors, and compare findings with that of controls. Method: IHC was applied using commercially available antibodies for ChAT, AChE, and BuChE on 7µm FFPE brain tissue from infants and piglets to optimise the stain by manipulating our standard IHC protocol and applying alternative detection systems. Once optimised, tissue sections at 3 levels of the medulla (caudal, intermediate, and rostral), from 45 infants categorised as explained SUDI (eSUDI, n=10), SIDS I (n=9), and SIDS II (n=26), were stained. Microscopic analysis and manual neuron counting to determine the percent positive neurons relative to the total number of neurons, for each marker, were performed for the DMNV. Statistical analysis using SPSS, applied one-way ANOVA to test for significance (p value ≤ 0.05) between diagnostic groups, while Independent sample t-test was used for risk factor comparison, and Chi-square was used to test categorical data such as smoke exposure and sex. Results: For chapter 2, the focus was on AChE and BuChE, and positive staining was visible for these in FFPE infant medulla, pons, and cerebellum tissue, however, that for BuChE did not match reported literature. As such Chapter 3 was only focused on ChAT and AChE to determine diagnostic and risk factor effects. The percentage of ChAT containing neurons was lower in the rostral DMNV (p=0.03) of SIDS II compared to eSUDI. Lower percentage of ChAT- positive neurons was also found in SIDS male infants (p=0.018) at the intermediate DMNV, and in the caudal (p=0.04) and intermediate (p=0.009) DMNV of the CSE group (all infants regardless of diagnosis) as well as infants with an URTI (p=0.013). Conclusion: Cholinergic neurons of the DMNV are sensitive to extrinsic risk factors such as CSE and male gender than being specific to SIDS. Given the rostral DMNV role is predominantly of lung regulation, while that of the caudal and intermediate are of abdominal and/or cardiac regulation, this links up with the risk factors and their perceived organs of affect; URTI with the lungs and CSE with heart and abdomen.