Interruptible Demyelination in Avian Riboflavin Deficient Neuropathy

Interruptible Demyelination in Avian Riboflavin Deficient Neuropathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Interruptible Demyelination in Avian Riboflavin Deficient Neuropathy Zhao Cai This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3865311/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2024 Read the published version in Cell & Bioscience → Version 1 posted 3 You are reading this latest preprint version Abstract Background and aims : The evolution of demyelination in individual internodes remains unclear although it has been noticed the paranodal demyelination precedes internodal demyelination in neuropathies with diverse aetiologies. For therapeutic purpose, it is fundamental to know whether the demyelinating procedure in affected internodes can be interrupted. This study aimed to delineate the development of demyelination in individual internodes in avian riboflavin deficient neuropathy. Methods : Newborn broiler meat chickens were maintained either on a routine diet containing 5.0 mg/kg riboflavin, a riboflavin deficient diet containing 1.8 mg/kg riboflavin, or initially a riboflavin deficient diet for 11 days and then routine diet plus riboflavin repletion from day 12. Evolution of demyelination in individual internodes was analyzed by teased nerve fibre studies from day 11 to 21. Results : In riboflavin deficient chickens, demyelination was the predominant feature: it was mainly confined to the paranodal region at day 11; extended into internodal region, but less than half of the internodal length in most affected internodes at day 16; involved more than half or whole internode at day 21. In the internode undergoing demyelination, myelin degeneration of varying degrees was noticed in the cytoplasm of the Schwann cell wrapping the internode. Two days after riboflavin repletion, co-existence of remyelination and active demyelination within individual internodes was noticed. Remyelination together with preserved short original internodes was the characteristic feature 4 and 9 days after riboflavin repletion. Conclusion : Riboflavin repletion interrupts the progression from paranodal to internodal demyelination in riboflavin deficient chickens and promotes remyelination before complete internodal demyelination. riboflavin demyelination remyelination teased nerve fibre Schwann cell Figures Figure 1 Figure 2 Figure 3 Introduction Segmental demyelination, firstly described in lead neuropathy in guinea pig by Gombault in 1880, refers to myelin degeneration of a paranode (paranodal segmental demyelination) or an internode (internodal segmental demyelination) [ 10 ]. Early changes at the paranodal region have been found in demyelination of many types either primary, such as inflammatory [ 17 , 18 ] and toxic [ 1 , 26 , 27 ] or secondary to axonal change [ 11 ]. It is believed that paranodal demyelination precedes internodal demyelination in both the peripheral nervous system [ 4 ] and central nervous system [ 12 , 24 ]. However, the procedure from paranodal to complete internodal demyelination has not been previously described. For potential therapeutic purpose, it is fundamental to know whether the progression from paranodal to internodal demyelination is an irreversible process once initiated or whether the demyelinating procedure in affected internodes can be interrupted. Riboflavin (a water-soluble vitamin, vitamin B2, VB 2 ) and its derivatives are coenzymes for numerous oxidases and dehydrogenases in eukaryotic cells [ 21 ]. Riboflavin deficiency leads to impaired β-oxidation of fatty acids [ 22 ] and significant change in proteomic profiles [ 29 ]. New-born chickens fed with riboflavin-deficient (VB 2 − ) diet develop a demyelinating peripheral neuropathy [ 5 , 8 , 14 , 15 , 27 ]. Remyelination and spontaneous recovery after about 3 weeks may be due to endogenous microbial synthesis of riboflavin in the intestine (absent in newborn chicks) [ 25 ]. Previous studies in avian riboflavin deficient neuropathy found early changes at the paranodal region [ 8 , 14 ], implying beginning of this pathogenic process. Here, by way of teased nerve fibre (TF) studies, we delineated the evolution of demyelination in affected internodes with the focus on interruption of this demyelinating procedure by VB 2 repletion (VB2 + ). Materials and Methods The avian riboflavin deficiency model of demyelinating peripheral neuropathy has previously been described [ 8 ]. In the current study, newborn broiler meat chickens (Cobb 500, Cobb-Vantress Inc, Arkansas, USA) were maintained ad libitum on (i) a conventional diet containing 5.0 mg/kg riboflavin, control groups; (ii) a deficient diet containing 1.8 mg/kg riboflavin, VB 2 − groups; (iii) initial riboflavin deficient diet for 11 days and then from post hatch day 12 (PH12d) conventional diet plus intraperitoneal injection of 0.5 ml Vitamin B Complex Injection containing 0.5mg/ml VB2 (Ceva Animal Health, Australia), VB 2 −/+ groups. Five animals in each group were studied. Control and VB 2 − chickens were killed on PH11d, PH16d and PH21d, and VB 2 −/+ chickens on PH14d, PH16d and PH21d by transcardiac perfusion with 4% paraformaldehyde 0.1 M phosphate buffer (pH 7.4). Left sciatic and brachial nerves were processed according to a standard peripheral nerve biopsy protocol for light microscopy (LM) and electron microscopy (EM) [ 9 ]. Right sciatic and brachial nerves were prepared for sectional studies of resin teased nerve fibres (TFs) according to a published method [ 7 ]. In brief, after the perfusion, right sciatic and brachial nerves were further fixed in 4% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer for 1.5 h and postfixed in 1% osmium tetroxide for 2 h. After washing in the cacodylate buffer, nerves were dehydrated in graded ethanol, softened in fresh epoxy resin for 3 days. Individual nerve fibres were isolated in fresh epoxy resin and mounted onto a carbon-coated slide. Fifty TFs from each nerve were prepared. Some fibres were further marked at specific sites using short TFs as “marker” fibres and left in an oven for resin polymerization. A small capsule (for routine electron microscopy resin embedding) filled with fresh resin was placed upside down onto the carbon-slide, covering the area that contained the teased fibres, and left in the oven for resin polymerization. The under surface of the slide was placed on top of a solid metal block, which was pre-cooled in liquid nitrogen. Due to the temperature difference, the resin block separated from the slide along the line of the carbon coat, with the fibres remaining on the surface of the resin block. Longitudinal sections of individual nerve fibres were collected from this resin block using an ultramicrotome. This resin block may also be trimmed around the fibres and placed in a capsule filled with fresh resin, keeping the teased fibres parallel to the long axis of the capsule, and the resin polymerized as above. The fibres were then transversely sectioned at specified sites to correlate with abnormalities seen on teased preparation. Results As reported previously, no neurological signs and pathological changes were found in control animals; VB 2 − chickens showed progressive weakness and paresis from PH8d until end of this study [ 8 ]. The paresis in VB 2 −/+ chickens became stable at PH16d (4 days after VB 2 repletion), and gradually recovered thereafter. Routine pathological examination revealed predominant demyelination in all VB 2 − groups and predominant remyelination in VB 2 −/+ chickens at PH16d and PH21d. Endoneurial oedema, hypertrophic Schwann cells with lipid deposition, complex myelin foldings with varying stages of degeneration and myelin fragments in Schwann cell cytoplasm, as noticed in VB 2 − chickens [ 8 ], were also found in VB 2 −/+ chickens at less severity. But there were no obvious difference of hypertrophic fibroblasts and onion bulb formation [ 5 ] between VB 2 − and VB 2 −/+ chickens at the same time points. Macrophage-mediated myelin stripping, as described in both human and animal inflammatory demyelinating neuropathies [ 3 , 18 ], was not found here. Sciatic and brachial nerves showed same pathological changes. TF studies in VB 2 − chickens Demyelination was the predominant change in TF preparations. In order to analyze the development of demyelination in affected internodes, here demyelination is arbitrarily divided into 3 groups: paranodal demyelination, partial internodal demyelination and internodal demyelination (Table 1 ). Quantitative analyses revealed: predominant paranodal demyelination at PH11d (Fig. 1 A), predominant partial internodal demyelination at PH16d (Fig. 1 B) and predominant internodal demyelination at PH21d (Fig. 1 C). In addition to previously reported enveloping fibroblastic reaction [ 5 ], sectional studies of 50 TFs with different types of demyelination revealed the following changes: (i) incipient myelin degeneration at the paranodal region; (ii) active myelin degeneration in myelin retained segment, more severe close to the denuded site; (iii) Schwann cell nucleus in the middle of myelin retained segment; (iv) myelin fragments in the cytoplasm of the Schwann cell enwrapping the internode; (v) preserved but attenuated axon in the demyelinated region and (vi) presence of different types of demyelination in the same TF preparation (Fig. 1 ). Table 1 Frequency of different types of demyelination in VB 2 − chickens Paranodal demyelination Partial internodal demyelination Internodal demyelination PH11d 84 ± 5.48 16 ± 5.48 0 PH16d 19 ± 2.65** 62.8 ± 4.66* 18.2 ± 5.22 PH21d 5.6 ± 1.51** 15.4 ± 3.91** 79 ± 3.54* Paranodal demyelination: demyelination affecting paranodal region, covering not more than 10 percent of the internodal length. Partial internodal demyelination: demyelination involving both paranodal and adjoining internodal region, covering more than 10 percent of internodal length and the demyelinated segment not longer than that of neighbouring myelin sheath retained segment. Internodal demyelination: demyelination affecting whole internode or the demyelinated segment longer than that of neighbouring myelin retained segment. ANOVA and student t tests revealed significantly higher (*) or lower (**) than that at the previous time point (P < 0.05) TF studies in VB 2 −/+ chickens At PH14d (2 days after riboflavin repletion), paranodal and partial internodal demyelination were the predominant change according to the surface appearances. But sectional studies revealed co-existence of remyelination and active demyelination within affected internodes in 14/20 such “demyelinating” fibres (Figs. 2 and 3 A): (i) Schwann cell nuclei in the “denuded” regions, even a very short segment (50µm) and a few numbers of myelin lamellae around the axon with inner mesaxon connecting the axon and outer mesaxon connecting the Schwann cell basal membrane, indicating this Schwann cell was myelinating the axon; (ii) active myelin degeneration at the peripheries of myelin retained segments (here named original internodes). At PH16d and PH21d (4 and 9 days after riboflavin repletion), remyelination (thin myelin sheath) was present in majority of TFs. In these remyelinating fibres, the characteristic feature was the short original internodes (with thick myelin sheaths), especially at PH21d: they were usually irregular, the shortest less than half of the longest and even some remyelinating internodes (Fig. 3 B); sometimes regular, similar to the length of adjacent remyelinating internodes (Fig. 3 C). Sectional studies of 38 such fibres revealed: (i) new nodes of Ranvier, which were hardly visible from the surface appearance especially at PH16d; (ii) active myelin degeneration at the peripheries of original internodes at PH16d, but much less in frequency and severity at PH21d; (iii) myelin debris in the cytoplasm of myelinating Schwann cell encompassing the original and sometimes remyelinating internodes. Enveloping fibroblasts as seen in VB2 − animals were also present in VB2 −/+ animals (Figs. 2 and 3 ). Discussion Demyelination in VB2 − chickens proceeds from paranodal to central internodal region In the current study, morphometric analyses revealed significant changes from predominant paranodal demyelination at PH11d, to predominant partial internodal demyelination at PH16d, and then predominant internodal demyelination at PH21d. Sectional studies of TFs further demonstrated incipient myelin degeneration at paranodal and later at internodal region. These findings confirmed that, as expected, demyelination in VB 2 − chickens proceeds from paranodal to internodal region until the full length of the internode is affected. Macrophage-mediated myelin stripping was not found in the current and previous studies by electron microscopy and immunohistochemistry. In contrast, varying stages of degenerated myelin fragments were found in the cytoplasm of hypertrophic Schwann cells [ 5 , 8 , 14 , 15 ], consistent with the myelin phagocytosis and clearance function of Schwann cell [ 2 , 13 ]. Sectional studies of TFs here proved that the Schwann cell expressing such myelinolytic activity was the same Schwann cell enwrapping the internode, indicating that the original myelinating Schwann cell plays a critical role in initiating and promoting demyelination within the same internode. Such Schwann cell is named demyelinating Schwann cell [ 19 ]. The longitudinal extension of myelin degeneration and clearance from paranodal to internodal region by demyelinating Schwann cells is named Demyelination Driven by Demyelinating Schwann Cell (DDDSC), which is thought the common pathway of demyelination in immune-independent and some inflammatory demyelinating neuropathies [ 19 , 20 ]. Interruptible demyelination and remyelination in VB2 −/+ chickens Although there has been no previous study specifying the possibility to interrupt the evolution from paranodal to internodal demyelination, it is believed that whenever initiated, DDDSC will progress unidirectionally toward complete removal of intracellular myelin sheath debris before remyelination happens. This process is so-called irreversible demyelination [ 19 ]. But in the notion of irreversible demyelination [ 19 ], two facts are overlooked, rapid progression of the disease and continuous, even irreversible, effect of the pathogen. As the progression from paranodal to complete internodal demyelination takes a few (5–10) days in the current avian model, it provides a time window to interfere with this procedure. Importantly, the pathogenic factor, VB 2 − , is readily rectified by VB 2 + . VB 2 + was applied from PH12d as myelin degeneration in most affected internodes was limited to the paranodal and adjoining regions with preservation of central internodal myelin segments at this time. Co-existence of remyelination and active demyelination within individual internodes in VB 2 −/+ PH14d chickens indicates occurrence of remyelination before complete internodal demyelination. Presence of short original internodes in remyelinating fibres, especially at PH21d, suggests the demyelinating procedure in affected internodes be interrupted. This type of demyelination and remyelination in avian riboflavin deficiency is here named as interruptible demyelination driven by Schwann cell (IDDSC). Whether IDDSC applies to avian riboflavin deficiency only? The morphological features, myelin debris in Schwann cell cytoplasm, intercalated internode and so called “hypomyelination” noticed by conventional pathological examinations [ 10 , 16 , 23 ], suggest that IDDSC may apply to demyelinating neuropathies with diverse aetiologies. Further studies may prove this speculation. In conclusion, demyelination in avian VB 2 − neuropathy proceeds from paranodal gradually to internode region; the Schwann cell, originally myelinating the internode, plays a pivotal role in this demyelinating procedure, myelin degeneration and clearance; VB 2 + interrupts the demyelinating procedure in affected internodes and promotes remyelination before complete internodal demyelination. Declarations Ethics approval: This experimental protocol was approved by the Animal Ethics Committee of the Institute of Medical and Veterinary Science (now named SA Pathology), Adelaide and conformed to the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose. Consent for publication: The author and the affiliated organizations give full consent for publication of this manuscript. Availability of data and materials : The data and materials are available. Competing interests : The author does not have competing interests. Funding : This research was initially supported by the Department funding. Because of the time-consuming nature of the specific technique, which is essential for this study, the funding was run out before the research is completed. Currently there is no public funding for it. Author’s contributions : The sole author contribute fully to this manuscript. Acknowledgments: The author thanks for long supports and valuable opinions for this manuscript from professors Peter C Blumbergs and John C Finnie and Mr Jim Manavis. Thanks to Inghams Enterprises, Adelaide, for the support of this study. References Allt G, Cavanagh JB. Ultrastructural changes in the region of the node of Ranvier in the rat caused by diphtheria toxin. Brain. 1969;92:459–68. https://doi.org/10.1093/brain/92.2.459 . Balakrishnan A, Belfiore L, Chu TH, et al. Insights into the role and potential of Schwann Cells for peripheral nerve repair from studies of development and injury. Front Mol Neurosci. 2021;13:608442. https://doi.org/10.3389/fnmol.2020.608442 . Bensfield AC, Evans J, Pesayco JP, et al. Recurrent demyelination and remyelination in 37 young Bengal cats with polyneuropathy. J Vet Intern Med. 2011;25:882–9. https://doi.org/10.1111/j.1939-1676.2011.0740.x . Bilbao JM, Schmidt RE. (2015) Biopsy Diagnosis of Peripheral Neuropathy. 2nd edn. pp 85–110. 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Riboflavin deficiency induces a significant change in proteomic profiles in HepG2 cells. Sci Rep. 2017;7:45861. 10.1038/srep45861 . Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2024 Read the published version in Cell & Bioscience → Version 1 posted Reviewers invited by journal 28 Jan, 2024 Editor assigned by journal 24 Jan, 2024 First submitted to journal 21 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3865311","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269842769,"identity":"53991257-fa8a-4164-9768-61b0d27dccfd","order_by":0,"name":"Zhao Cai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYDACCQjFw0+6FskGUrUwGBwgVof87OaHDxhq7GSMzx9+wPil4jAD/4wE/FoY5xwzNmA4lsxjdiPNgFnmzGEGiRsEtDBLJJhJMDYwA7UwGDBLtqUxMBDSwiaR/g2opZ7HuP/4B7AWeUJaeCRyQLYc5jFgyDFg/Nhmw2BASIuERE6xQcKx4zwSN3IKDjOcseExPPMAvxb5GekbH3yoqbbn7z++8eGPCgk5ueMEbAEDmJrDPECXEqEeCTD+IE39KBgFo2AUjBAAAPcrO03kKK/lAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6564-0592","institution":"SA Pathology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Cai","suffix":""}],"badges":[],"createdAt":"2024-01-15 03:42:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3865311/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3865311/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13578-024-01233-5","type":"published","date":"2024-04-22T00:24:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50447153,"identity":"a6e70e23-e587-45e6-9701-5a4db5ee6fac","added_by":"auto","created_at":"2024-01-31 16:18:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":522957,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of demyelination in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003e chickens. Light micrographs. \u003cstrong\u003eA\u003c/strong\u003e: Two upper panels show part of a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003ePH11d chicken. The nodes of Ranvier are indicated by arrows (N\u003csub\u003e1\u003c/sub\u003e to N\u003csub\u003e5\u003c/sub\u003e). Paranodal demyelination is noted at 2 sites (N\u003csub\u003e3\u003c/sub\u003e and N\u003csub\u003e4\u003c/sub\u003e). Schwann cell nuclei (white arrowheads) are located in the middle of each internode. Panels 3 to 5 are longitudinal sections through designated segments. Panel 3 is through the first paranodal demyelinating area. No discernible myelin sheath is seen in the demyelinated segment. Myelin degeneration (large arrowheads in panel 3) is seen in the internodal region adjoining the paranodal demyelination (small arrowheads in panel 2). Panel 4 is through the central part of the internode with paranodal demyelination. The myelin sheath is well preserved in this segment and a Schwann cell nucleus (SN) is present. Panel 5 is through the second paranodal demyelinating area. No discernible myelin sheath is seen in the demyelinated segment. Myelin degeneration (large arrowheads in panel 5) is seen in longitudinal section through the segment (small arrowheads in panel 2) adjoining the paranodal demyelination. The axon is attenuated in demyelinating regions. \u003cstrong\u003eB \u003c/strong\u003e(adapted from Cai et al., 2007 [5] with permission from the author and publisher): The three upper panels show a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003ePH16d chicken displaying paranodal demyelination (N\u003csub\u003e1\u003c/sub\u003e and N\u003csub\u003e4\u003c/sub\u003e) and partial internodal demyelination (N\u003csub\u003e2\u003c/sub\u003e, and N\u003csub\u003e3\u003c/sub\u003e, N\u003csub\u003e5\u003c/sub\u003e and N\u003csub\u003e6\u003c/sub\u003e). Paranodal swellings (small arrowheads) and surrounding pale-osmicated material representing the fibroblastic onion bulb like proliferation [5]. Panels 4 to 6 are longitudinal sections through designated segments in the three upper panels. In the demyelinated segments, there is no discernible myelin sheath and axon (a) is attenuated. Redundant myelin foldings with varying degrees of myelin splitting and degeneration (large arrowheads) are found in longitudinal sections through paranodal swellings while the myelin sheaths in the neighbouring non-demyelinated internodal regions are intact. The fibre is surrounded by a variably thick fibroblastic proliferation consisting of fibroblast processes and collagen fibres. Numerous fibroblast nuclei (arrows), some with multiple distinct nucleoli (double arrows), are noted at both demyelinated and non-demyelinated segments. \u003cstrong\u003eC\u003c/strong\u003e: Tow upper panels show part of a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−\u003c/sup\u003ePH21d chicken displaying internodal demyelination. Two lower panels are cross sections through designated sites of the TF, showing denuded axon in the demyelinated segments (L1, 5-19), redundant myelin foldings with myelin splitting and degeneration at both ends of the myelin-maintained segment (L2 and 4). The axon size in the region with intact myelin (L3) is larger than that of demyelinated regions. Fibroblast processes and collagen ensheath the TF at all levels to some degree.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3865311/v1/318d44d2c88825c13e862020.png"},{"id":50447829,"identity":"ac49052a-cf5d-459d-ada1-e735fab91c77","added_by":"auto","created_at":"2024-01-31 16:26:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":393815,"visible":true,"origin":"","legend":"\u003cp\u003eLight micrographs, panels 1 and 3; Electron micrographs, panels 2 and 4. The first panel is part of a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−/+\u003c/sup\u003ePH14d chicken displaying paranodal and partial internodal demyelination according to the surface appearance. Panel 2 is a longitudinal section through the paranodal demyelination segment. A Schwann cell nucleus (SN) is present. This Schwann cell envelops the axon under basal lamella of the nerve fibre (inserts). Panel 3 left is a longitudinal section through the middle of the neighbouring myelin-maintained segment showing a Schwann cell nucleus (SN), 2 attached fibroblasts (Fi) and myelin debris (white arrows). Panel 3 right is a longitudinal section through the partial internodal demyelination segment. A centrally located Schwann cell nucleus (SN) is present. Redundant myelin foldings with myelin breakdown (white arrows) is present in the paranodal region of the neighbouring myelin-maintained segment. Panel 4 is a longitudinal section through the partial internodal demyelination region (arrowhead in panel 1) showing 4 layers of non-compacted myelin lamellae surrounding the axon under the basal lamella of the nerve fibre: an inner mesaxon (white arrowhead) connecting the axolemma and outer mesaxon (double white arrowheads) connecting Schwann cell basement membrane.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3865311/v1/14f8ad27bafb4fab1e302371.png"},{"id":50447152,"identity":"0d293fe2-490d-4de5-a951-da68c9ec4b80","added_by":"auto","created_at":"2024-01-31 16:18:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":485326,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of demyelination and remyelination in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−/+\u003c/sup\u003e chickens. Light micrographs except for electron micrograph at panel 6 in \u003cstrong\u003eA\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e: Panels 1 and 2 are part of a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−/+\u003c/sup\u003ePH14d chicken with partial internodal demyelination. Panels 3 to 5 are longitudinal sections through designated sites of the TF. Redundant myelin foldings are seen in externally normal paranodal regions (panel 3 left) and paranodal regions adjoining partial internodal demyelination (panels 4 and 5). A centrally located Schwann cell nucleus (SN) is noticed in the demyelinated regions (panels 4 and 5) and neighbouring myelin-maintained segment (panel 3 right). Supernumerary fibroblasts (Fi) are seen attached to the TF preparation at paranodal and internodal regions. Electron microscopy (panel 6) further demonstrates the fibroblast with enriched rough reticulum (white rectangle in panel 3 left). \u003cstrong\u003eB\u003c/strong\u003e: Two upper panels are part of a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−/+\u003c/sup\u003ePH16d chicken, showing remyelination. Three lower panels are longitudinal sections of designated segments. The nodes of Ranvier (N\u003csub\u003e1\u003c/sub\u003e-N\u003csub\u003e13\u003c/sub\u003e arrows) are hardly seen from the surface appearance of the TF, but are clearly identified in longitudinal sections. The 1\u003csup\u003est\u003c/sup\u003e (N\u003csub\u003e1\u003c/sub\u003e-N\u003csub\u003e2\u003c/sub\u003e), 4\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e4\u003c/sub\u003e-N\u003csub\u003e5\u003c/sub\u003e), 8\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e8\u003c/sub\u003e-N\u003csub\u003e9\u003c/sub\u003e) and 11\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e11\u003c/sub\u003e-N\u003csub\u003e12\u003c/sub\u003e) internodes are original internodes with thick myelin sheaths. The 2\u003csup\u003end\u003c/sup\u003e (N\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e3\u003c/sub\u003e), 3\u003csup\u003erd\u003c/sup\u003e (N\u003csub\u003e3\u003c/sub\u003e-N\u003csub\u003e4\u003c/sub\u003e), 5\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e5\u003c/sub\u003e-N\u003csub\u003e6\u003c/sub\u003e), 6\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e6\u003c/sub\u003e-N\u003csub\u003e7\u003c/sub\u003e), 7\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e7\u003c/sub\u003e-N\u003csub\u003e8\u003c/sub\u003e), 9\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e9\u003c/sub\u003e-N\u003csub\u003e10\u003c/sub\u003e), 10\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e10\u003c/sub\u003e-N\u003csub\u003e11\u003c/sub\u003e) and 12\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e12\u003c/sub\u003e-N\u003csub\u003e13\u003c/sub\u003e) internodes are remyelinating internodes with thin myelin sheaths. Substantial variation of the internodal length is seen in both original and remyelinating internodes. The length of some remyelinating internodes, such as the 2\u003csup\u003end\u003c/sup\u003e (N\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e3\u003c/sub\u003e), 3\u003csup\u003erd\u003c/sup\u003e (N\u003csub\u003e3\u003c/sub\u003e-N\u003csub\u003e4\u003c/sub\u003e), 7\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e7\u003c/sub\u003e-N\u003csub\u003e8\u003c/sub\u003e) and 10\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e10\u003c/sub\u003e-N\u003csub\u003e11\u003c/sub\u003e) internodes, are similar or even longer than some original internodes, such as the 4\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e4\u003c/sub\u003e-N\u003csub\u003e5\u003c/sub\u003e) and 8\u003csup\u003eth\u003c/sup\u003e (N\u003csub\u003e8\u003c/sub\u003e-N\u003csub\u003e9\u003c/sub\u003e) internodes. Focal myelin swellings (arrowheads) are present at the paranodal regions of original internodes. The nodal gap is not extra ordinarily large, implicating that the length of the remyelinating internode is already fixed at this stage. A Schwann cell nucleus (SN) and lipid deposition (asterisk) are present in the middle of an original internode. Myelin debris is seen in the paranodal Schwann cell cytoplasm of the original internode (double arrows). The TF is surrounded by a variably thick fibroblastic proliferation consisting of fibroblast processes and collagen fibres. Numerous fibroblast nuclei (white arrows) are noted in the longitudinal section at both original and remyelinating internodes. \u003cstrong\u003eC\u003c/strong\u003e: The first panel is part of a TF from a VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e−/+\u003c/sup\u003ePH21d chicken, showing remyelination. The nodes of Ranvier are indicated by arrows (N\u003csub\u003e1\u003c/sub\u003e-N\u003csub\u003e6\u003c/sub\u003e). There is no considerable variation of the internodal length between the original (N\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e3\u003c/sub\u003e and N\u003csub\u003e4\u003c/sub\u003e-N\u003csub\u003e5\u003c/sub\u003e) and remyelinating internodes (N\u003csub\u003e1\u003c/sub\u003e-N\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e3\u003c/sub\u003e-N\u003csub\u003e4\u003c/sub\u003e and N\u003csub\u003e5\u003c/sub\u003e-N\u003csub\u003e6\u003c/sub\u003e). One of the remyelinating internode (N\u003csub\u003e1\u003c/sub\u003e-N\u003csub\u003e2\u003c/sub\u003e, 194 µm in length) is even longer than that of original internodes (N\u003csub\u003e2\u003c/sub\u003e-N\u003csub\u003e3\u003c/sub\u003e, 176 µm; N\u003csub\u003e4\u003c/sub\u003e-N\u003csub\u003e5\u003c/sub\u003e, 159 µm). Panels 2 and 3 are longitudinal sections through the full length of a remyelinating and an original internode in the first panel. Schwann cell nucleus (SN) is present in the middle of both the remyelinating and original internodes. Degenerating myelin of varying stages (double arrows) is noticed in Schwann cell cytoplasm of both original and remyelinating internodes. White arrows indicate fibroblast nuclei.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3865311/v1/bef88c837c8a042a00eefee7.png"},{"id":55697134,"identity":"14e6fab4-be58-4656-b9c1-9c04c82ec1ff","added_by":"auto","created_at":"2024-05-02 02:09:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1874993,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865311/v1/add813c9-4e18-4cfc-8803-92990b3a4b2d.pdf"}],"financialInterests":"","formattedTitle":"Interruptible Demyelination in Avian Riboflavin Deficient Neuropathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSegmental demyelination, firstly described in lead neuropathy in guinea pig by Gombault in 1880, refers to myelin degeneration of a paranode (paranodal segmental demyelination) or an internode (internodal segmental demyelination) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Early changes at the paranodal region have been found in demyelination of many types either primary, such as inflammatory [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and toxic [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] or secondary to axonal change [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is believed that paranodal demyelination precedes internodal demyelination in both the peripheral nervous system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and central nervous system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the procedure from paranodal to complete internodal demyelination has not been previously described. For potential therapeutic purpose, it is fundamental to know whether the progression from paranodal to internodal demyelination is an irreversible process once initiated or whether the demyelinating procedure in affected internodes can be interrupted.\u003c/p\u003e \u003cp\u003eRiboflavin (a water-soluble vitamin, vitamin B2, VB\u003csub\u003e2\u003c/sub\u003e) and its derivatives are coenzymes for numerous oxidases and dehydrogenases in eukaryotic cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Riboflavin deficiency leads to impaired β-oxidation of fatty acids [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and significant change in proteomic profiles [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. New-born chickens fed with riboflavin-deficient (VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) diet develop a demyelinating peripheral neuropathy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Remyelination and spontaneous recovery after about 3 weeks may be due to endogenous microbial synthesis of riboflavin in the intestine (absent in newborn chicks) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous studies in avian riboflavin deficient neuropathy found early changes at the paranodal region [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], implying beginning of this pathogenic process. Here, by way of teased nerve fibre (TF) studies, we delineated the evolution of demyelination in affected internodes with the focus on interruption of this demyelinating procedure by VB\u003csub\u003e2\u003c/sub\u003e repletion (VB2\u003csup\u003e+\u003c/sup\u003e).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe avian riboflavin deficiency model of demyelinating peripheral neuropathy has previously been described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the current study, newborn broiler meat chickens (Cobb 500, Cobb-Vantress Inc, Arkansas, USA) were maintained \u003cem\u003ead libitum\u003c/em\u003e on (i) a conventional diet containing 5.0 mg/kg riboflavin, control groups; (ii) a deficient diet containing 1.8 mg/kg riboflavin, VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e groups; (iii) initial riboflavin deficient diet for 11 days and then from post hatch day 12 (PH12d) conventional diet plus intraperitoneal injection of 0.5 ml Vitamin B Complex Injection containing 0.5mg/ml VB2 (Ceva Animal Health, Australia), VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e groups. Five animals in each group were studied. Control and VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens were killed on PH11d, PH16d and PH21d, and VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens on PH14d, PH16d and PH21d by transcardiac perfusion with 4% paraformaldehyde 0.1 M phosphate buffer (pH 7.4). Left sciatic and brachial nerves were processed according to a standard peripheral nerve biopsy protocol for light microscopy (LM) and electron microscopy (EM) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Right sciatic and brachial nerves were prepared for sectional studies of resin teased nerve fibres (TFs) according to a published method [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In brief, after the perfusion, right sciatic and brachial nerves were further fixed in 4% paraformaldehyde/2.5% glutaraldehyde in 0.1 M phosphate buffer for 1.5 h and postfixed in 1% osmium tetroxide for 2 h. After washing in the cacodylate buffer, nerves were dehydrated in graded ethanol, softened in fresh epoxy resin for 3 days. Individual nerve fibres were isolated in fresh epoxy resin and mounted onto a carbon-coated slide. Fifty TFs from each nerve were prepared. Some fibres were further marked at specific sites using short TFs as \u0026ldquo;marker\u0026rdquo; fibres and left in an oven for resin polymerization. A small capsule (for routine electron microscopy resin embedding) filled with fresh resin was placed upside down onto the carbon-slide, covering the area that contained the teased fibres, and left in the oven for resin polymerization. The under surface of the slide was placed on top of a solid metal block, which was pre-cooled in liquid nitrogen. Due to the temperature difference, the resin block separated from the slide along the line of the carbon coat, with the fibres remaining on the surface of the resin block. Longitudinal sections of individual nerve fibres were collected from this resin block using an ultramicrotome. This resin block may also be trimmed around the fibres and placed in a capsule filled with fresh resin, keeping the teased fibres parallel to the long axis of the capsule, and the resin polymerized as above. The fibres were then transversely sectioned at specified sites to correlate with abnormalities seen on teased preparation.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAs reported previously, no neurological signs and pathological changes were found in control animals; VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens showed progressive weakness and paresis from PH8d until end of this study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The paresis in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens became stable at PH16d (4 days after VB\u003csub\u003e2\u003c/sub\u003e repletion), and gradually recovered thereafter. Routine pathological examination revealed predominant demyelination in all VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e groups and predominant remyelination in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens at PH16d and PH21d. Endoneurial oedema, hypertrophic Schwann cells with lipid deposition, complex myelin foldings with varying stages of degeneration and myelin fragments in Schwann cell cytoplasm, as noticed in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], were also found in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens at less severity. But there were no obvious difference of hypertrophic fibroblasts and onion bulb formation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] between VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens at the same time points. Macrophage-mediated myelin stripping, as described in both human and animal inflammatory demyelinating neuropathies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], was not found here. Sciatic and brachial nerves showed same pathological changes.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTF studies in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens\u003c/h2\u003e \u003cp\u003eDemyelination was the predominant change in TF preparations. In order to analyze the development of demyelination in affected internodes, here demyelination is arbitrarily divided into 3 groups: paranodal demyelination, partial internodal demyelination and internodal demyelination (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Quantitative analyses revealed: predominant paranodal demyelination at PH11d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), predominant partial internodal demyelination at PH16d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and predominant internodal demyelination at PH21d (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition to previously reported enveloping fibroblastic reaction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], sectional studies of 50 TFs with different types of demyelination revealed the following changes: (i) incipient myelin degeneration at the paranodal region; (ii) active myelin degeneration in myelin retained segment, more severe close to the denuded site; (iii) Schwann cell nucleus in the middle of myelin retained segment; (iv) myelin fragments in the cytoplasm of the Schwann cell enwrapping the internode; (v) preserved but attenuated axon in the demyelinated region and (vi) presence of different types of demyelination in the same TF preparation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency of different types of demyelination in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParanodal demyelination\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePartial internodal demyelination\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInternodal demyelination\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePH11d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e84\u0026thinsp;\u0026plusmn;\u0026thinsp;5.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;5.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePH16d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e62.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.66*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePH21d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e79\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eParanodal demyelination: demyelination affecting paranodal region, covering not more than 10 percent of the internodal length.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003ePartial internodal demyelination: demyelination involving both paranodal and adjoining internodal region, covering more than 10 percent of internodal length and the demyelinated segment not longer than that of neighbouring myelin sheath retained segment.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eInternodal demyelination: demyelination affecting whole internode or the demyelinated segment longer than that of neighbouring myelin retained segment.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eANOVA and student \u003cem\u003et\u003c/em\u003e tests revealed significantly higher (*) or lower (**) than that at the previous time point (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTF studies in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens\u003c/h2\u003e \u003cp\u003eAt PH14d (2 days after riboflavin repletion), paranodal and partial internodal demyelination were the predominant change according to the surface appearances. But sectional studies revealed co-existence of remyelination and active demyelination within affected internodes in 14/20 such \u0026ldquo;demyelinating\u0026rdquo; fibres (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA): (i) Schwann cell nuclei in the \u0026ldquo;denuded\u0026rdquo; regions, even a very short segment (50\u0026micro;m) and a few numbers of myelin lamellae around the axon with inner mesaxon connecting the axon and outer mesaxon connecting the Schwann cell basal membrane, indicating this Schwann cell was myelinating the axon; (ii) active myelin degeneration at the peripheries of myelin retained segments (here named original internodes).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt PH16d and PH21d (4 and 9 days after riboflavin repletion), remyelination (thin myelin sheath) was present in majority of TFs. In these remyelinating fibres, the characteristic feature was the short original internodes (with thick myelin sheaths), especially at PH21d: they were usually irregular, the shortest less than half of the longest and even some remyelinating internodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); sometimes regular, similar to the length of adjacent remyelinating internodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Sectional studies of 38 such fibres revealed: (i) new nodes of Ranvier, which were hardly visible from the surface appearance especially at PH16d; (ii) active myelin degeneration at the peripheries of original internodes at PH16d, but much less in frequency and severity at PH21d; (iii) myelin debris in the cytoplasm of myelinating Schwann cell encompassing the original and sometimes remyelinating internodes. Enveloping fibroblasts as seen in VB2\u003csup\u003e\u0026minus;\u003c/sup\u003e animals were also present in VB2\u003csup\u003e\u0026minus;/+\u003c/sup\u003e animals (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDemyelination in VB2\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens proceeds from paranodal to central internodal region\u003c/h2\u003e \u003cp\u003eIn the current study, morphometric analyses revealed significant changes from predominant paranodal demyelination at PH11d, to predominant partial internodal demyelination at PH16d, and then predominant internodal demyelination at PH21d. Sectional studies of TFs further demonstrated incipient myelin degeneration at paranodal and later at internodal region. These findings confirmed that, as expected, demyelination in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e chickens proceeds from paranodal to internodal region until the full length of the internode is affected. Macrophage-mediated myelin stripping was not found in the current and previous studies by electron microscopy and immunohistochemistry. In contrast, varying stages of degenerated myelin fragments were found in the cytoplasm of hypertrophic Schwann cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], consistent with the myelin phagocytosis and clearance function of Schwann cell [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Sectional studies of TFs here proved that the Schwann cell expressing such myelinolytic activity was the same Schwann cell enwrapping the internode, indicating that the original myelinating Schwann cell plays a critical role in initiating and promoting demyelination within the same internode. Such Schwann cell is named demyelinating Schwann cell [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The longitudinal extension of myelin degeneration and clearance from paranodal to internodal region by demyelinating Schwann cells is named Demyelination Driven by Demyelinating Schwann Cell (DDDSC), which is thought the common pathway of demyelination in immune-independent and some inflammatory demyelinating neuropathies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInterruptible demyelination and remyelination in VB2\u003csup\u003e\u0026minus;/+\u003c/sup\u003e chickens\u003c/h2\u003e \u003cp\u003eAlthough there has been no previous study specifying the possibility to interrupt the evolution from paranodal to internodal demyelination, it is believed that whenever initiated, DDDSC will progress unidirectionally toward complete removal of intracellular myelin sheath debris before remyelination happens. This process is so-called irreversible demyelination [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. But in the notion of irreversible demyelination [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], two facts are overlooked, rapid progression of the disease and continuous, even irreversible, effect of the pathogen. As the progression from paranodal to complete internodal demyelination takes a few (5\u0026ndash;10) days in the current avian model, it provides a time window to interfere with this procedure. Importantly, the pathogenic factor, VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, is readily rectified by VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e was applied from PH12d as myelin degeneration in most affected internodes was limited to the paranodal and adjoining regions with preservation of central internodal myelin segments at this time. Co-existence of remyelination and active demyelination within individual internodes in VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;/+\u003c/sup\u003ePH14d chickens indicates occurrence of remyelination before complete internodal demyelination. Presence of short original internodes in remyelinating fibres, especially at PH21d, suggests the demyelinating procedure in affected internodes be interrupted. This type of demyelination and remyelination in avian riboflavin deficiency is here named as interruptible demyelination driven by Schwann cell (IDDSC). Whether IDDSC applies to avian riboflavin deficiency only? The morphological features, myelin debris in Schwann cell cytoplasm, intercalated internode and so called \u0026ldquo;hypomyelination\u0026rdquo; noticed by conventional pathological examinations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], suggest that IDDSC may apply to demyelinating neuropathies with diverse aetiologies. Further studies may prove this speculation.\u003c/p\u003e \u003cp\u003eIn conclusion, demyelination in avian VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e neuropathy proceeds from paranodal gradually to internode region; the Schwann cell, originally myelinating the internode, plays a pivotal role in this demyelinating procedure, myelin degeneration and clearance; VB\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e interrupts the demyelinating procedure in affected internodes and promotes remyelination before complete internodal demyelination.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e This experimental protocol was approved by the Animal Ethics Committee of the Institute of Medical and Veterinary Science (now named SA Pathology), Adelaide and conformed to the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e The author and the affiliated organizations give full consent for publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e: The data and materials are available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The author does not have competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This research was initially supported by the Department funding. Because of the time-consuming nature of the specific technique, which is essential for this study, the funding was run out before the research is completed. Currently there is no public funding for it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e: The sole author contribute fully to this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The author thanks for long supports and valuable opinions for this manuscript from professors Peter C Blumbergs and John C Finnie and Mr Jim Manavis. Thanks to Inghams Enterprises, Adelaide, for the support of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllt G, Cavanagh JB. Ultrastructural changes in the region of the node of Ranvier in the rat caused by diphtheria toxin. 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Sci Rep. 2017;7:45861. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep45861\u003c/span\u003e\u003cspan address=\"10.1038/srep45861\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"riboflavin, demyelination, remyelination, teased nerve fibre, Schwann cell","lastPublishedDoi":"10.21203/rs.3.rs-3865311/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3865311/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and aims\u003c/strong\u003e: The evolution of demyelination in individual internodes remains unclear although it has been noticed the paranodal demyelination precedes internodal demyelination in neuropathies with diverse aetiologies. For therapeutic purpose, it is fundamental to know whether the demyelinating procedure in affected internodes can be interrupted. This study aimed to delineate the development of demyelination in individual internodes in avian riboflavin deficient neuropathy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Newborn broiler meat chickens were maintained either on a routine diet containing 5.0 mg/kg riboflavin, a riboflavin deficient diet containing 1.8 mg/kg riboflavin, or initially a riboflavin deficient diet for 11 days and then routine diet plus riboflavin repletion from day 12. Evolution of demyelination in individual internodes was analyzed by teased nerve fibre studies from day 11 to 21.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: In riboflavin deficient chickens, demyelination was the predominant feature: it was mainly confined to the paranodal region at day 11; extended into internodal region, but less than half of the internodal length in most affected internodes at day 16; involved more than half or whole internode at day 21. In the internode undergoing demyelination, myelin degeneration of varying degrees was noticed in the cytoplasm of the Schwann cell wrapping the internode. Two days after riboflavin repletion, co-existence of remyelination and active demyelination within individual internodes was noticed. Remyelination together with preserved short original internodes was the characteristic feature 4 and 9 days after riboflavin repletion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Riboflavin repletion interrupts the progression from paranodal to internodal demyelination in riboflavin deficient chickens and promotes remyelination before complete internodal demyelination.\u003c/p\u003e","manuscriptTitle":"Interruptible Demyelination in Avian Riboflavin Deficient Neuropathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-31 16:17:57","doi":"10.21203/rs.3.rs-3865311/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2024-01-28T21:35:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-25T01:16:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell \u0026 Bioscience","date":"2024-01-22T01:24:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-bioscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbio","sideBox":"Learn more about [Cell \u0026 Bioscience](http://cellandbioscience.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cbio/default.aspx","title":"Cell \u0026 Bioscience","twitterHandle":"@OACellBiology","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6cef8569-43b6-48e0-b8f2-4a47c44aad89","owner":[],"postedDate":"January 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-02T00:24:08+00:00","versionOfRecord":{"articleIdentity":"rs-3865311","link":"https://doi.org/10.1186/s13578-024-01233-5","journal":{"identity":"cell-and-bioscience","isVorOnly":false,"title":"Cell \u0026 Bioscience"},"publishedOn":"2024-04-22 00:24:08","publishedOnDateReadable":"April 22nd, 2024"},"versionCreatedAt":"2024-01-31 16:17:57","video":"","vorDoi":"10.1186/s13578-024-01233-5","vorDoiUrl":"https://doi.org/10.1186/s13578-024-01233-5","workflowStages":[]},"version":"v1","identity":"rs-3865311","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3865311","identity":"rs-3865311","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}