162 Chapter 6 [239, 240]. SMI312 is a pan-axonal marker with a mixture of monoclonal antibodies directed against extensively phosphorylated axonal epitopes on neurofilaments M and H, and is often used as an axonal injury marker [241, 242]. Proteolipid protein (PLP) immunohistochemistry and luxol fast blue (LFB) staining are sensitive to assess myelin integrity [243]. Where PLP is generally used to distinguish white-grey matter borders and areas of demyelination [244], LFB colours lipoproteins of myelin sheaths and can be used to identify remyelination and myelin swelling [245]. This study aimed to explore potential microscopic disruptions in axonal and myelin integrity in narcolepsy type 1 using immunohistochemistry on postmortem human brain tissue. Building on prior DTI findings, we hypothesized lower axonal density throughout the brain (excluding the cerebellum) and compromised myelin structure in narcolepsy type 1. Materials & methods The Netherlands Brain Bank collected the postmortem human brain tissue that was used for this study from clinically diagnosed and neuropathologically confirmed donors in compliance with European ethical and legal guidelines [246, 247]. Postmortem brain tissue from four donors with narcolepsy type 1 were matched with two control groups of five donors (as the first had no midbrain sections available), each matched for potential confounding factors, including sex, age, postmortem delay, cerebral spinal fluid pH, Braak neurofibrilary tangle (NFT) stage [248], Braak Lewy body stage [249], the clock time and month of death and brain fixation duration. Narcolepsy type 1 donors 1-3 (for clinical-pathological information see Table 2) had a typical narcolepsy type 1 phenotype (with corresponding postmortem-verified hypocretin levels and cell counts) [250]. The clinical documentation of narcolepsy type 1 donor 4 (Table 2) showed that their symptoms had improved with disappearance of cataplexy after chronic exposure to opiates. A previous study using the same postmortem tissue has suggested that chronic opiate use may increase the number of hypocretin-producing neurons (19% of neurons were still present in this same individual) and hereby alleviate narcolepsy symptoms [125]. Sensitivity analyses excluding this atypical narcolepsy type 1 donor 4 were performed and included in Appendix A, Supplementary Tables 3-5. All donors with narcolepsy type 1 were diagnosed and treated by experienced neurologists/somnologists in our specialized sleep-wake clinic Stichting Epilepsie Instellingen Nederland (SEIN). All control donors were free from neurological disorders.
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