Regional Vulnerability to Demyelination: A Comparative Study of the Effects of LPC, LPS, and Combined Toxins in Corpus Callosum and Spinal Cord

Regional Vulnerability to Demyelination: A Comparative Study of the Effects of LPC, LPS, and Combined Toxins in Corpus Callosum and Spinal Cord | 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 Regional Vulnerability to Demyelination: A Comparative Study of the Effects of LPC, LPS, and Combined Toxins in Corpus Callosum and Spinal Cord Divya Goyal, Krutika H. Dobariya, Siddharth Raj, Piyush Maheshkar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8517400/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Focal demyelination refers to localized loss of myelin sheath surrounds the nerve fibers. It impairs the efficient transmission of nerve impulses results in neurological symptoms. Our goal is to investigate the differential effects of lysolecithin (LPC) and lipopolysaccharides (LPS) on demyelinating processes of central nervous system. We established a novel demyelinating model using a combination of LPC and LPS with their differential effects in the corpus callosum of brain and the thoracic region of the spinal cord. To confirm the pattern of demyelination, behavioural analysis was performed. Further, brain and spinal cord samples were collected at Day-post injection-1, 3, 7, and 28. The extent of demyelination and the presence of Nissl + cells were assessed using Luxol fast blue and Nissl staining respectively. Immunofluorescence studies were done to examine demyelination and its impact on endothelial cells dysfunction, astrogliosis, and activated microglia. Our data revealed that the combination group exhibited remarkable demyelination compared to the LPC and LPS groups alone. Additionally, vascular dysfunction, astrogliosis, and activated microglia were more pronounced in the combination group. Moreover, we found much of these effects were most promising in the spinal cord compared to corpus callosum, suggesting the presence of compensatory mechanisms and a unique brain microenvironment. This study is the first to demonstrate the integrated effects of LPC and LPS toxins compared to their individual effects in both the brain and spinal cord. Lipopolysaccharide Lysolecithin Spinal cord Focal demyelination Corpus callosum Remyelination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction In the central nervous system (CNS), myelin is a lipid-rich protective sheath that wraps around axons, facilitating communication between neurons by enabling rapid impulse propagation (Bercury and Macklin, 2015 ). The breakdown or loss of myelin leads to demyelination, a process associated with various neurodegenerative and pathological conditions, including multiple sclerosis (MS), Parkinson’s disease, Alzheimer’s disease, acute disseminated encephalomyelitis and traumatic injuries of the brain and spinal cord (Shi et al., 2015 ; Dong et al., 2018 ). Myelin constitutes the white matter within brain regions and surrounds the spinal cord periphery. When myelin is disrupted, it results in white matter loss, neuroinflammation, and deficits in motor behaviour (Sherman and Brophy, 2005 ; Popescu and Lucchinetti, 2012 ). Several in vivo models of demyelination have been established to understand this process. The most widely used is the experimental autoimmune encephalomyelitis (EAE) model, which is induced through immunization with antigens (Constantinescu et al., 2011 ; Glatigny and Bettelli, 2018 ), mimicking the autoimmune nature of MS, although it lacks the genetic and environmental factors contributing to demyelination (Melamed et al., 2022 ). The cuprizone model, a copper chelator, induces demyelination by impairing oligodendrocyte function (Torkildsen et al., 2008 ; Zirngibl et al., 2022 ), though it causes non-specific demyelination across various brain regions and is typically transient (Zirngibl et al., 2022 ). Alternatively, toxins-induced models, such as those using lysolecithin (LPC) and ethidium bromide, directly damage myelin without immune sensitization (Hollis et al., 2015 ; Plemel et al., 2018b ), primarily focusing on primary demyelination and targets myelinating cells. While such models lack the immune-mediated complexity of diseases like MS, they allow for a more controlled investigation of demyelination processes. LPC, integrates into the cellular membrane to increase permeability and disrupts myelin integrity, though it is short-lived and is cleared from white matter within 24 hours (Plemel et al., 2018a ). Conversely, LPS, a potent inflammatory mediator, binds to TLR4 receptors on microglia, triggering their activation and promoting the release of pro-inflammatory cytokines and chemokines, which drive neuroinflammation (Felts et al., 2005 ). The distinct mechanisms of LPC and LPS led us to explore a novel combined model. We hypothesized that the synergistic effects of these two toxins would produce a more consistent and robust demyelination and neuroinflammatory phenotype, potentially enhancing neurovascular and glial responses. By combining LPS and LPC, our goal is to establish a more comprehensive and clinically relevant model for studying demyelinating diseases. 2. MATERIALS AND METHODS 2.1 Animals Adult C57/BL6 male mice (Zydus, Ahmedabad, India), were used to carry out the experimental study. The mice, aged 6–8 weeks and weighing around 25–30 g, were randomly assigned into four groups: Sham, LPC, LPS, and LPC + LPS. Each group consisted of mice allocated for injection studies either in the spinal cord or corpus callosum. All animals were housed in a facility with controlled environmental conditions, maintaining humidity at 55–65%, temperature at 24 ± 3°C, and a 12-hour light-dark cycle. Food and water were provided ad libitum. All procedures and experiments were conducted in accordance with the protocol (IAEC/2023/032) approved by the Institutional Animal Ethics Committee of NIPER-Ahmedabad and the principles of laboratory animal care. 2.2 Induction of focal demyelination in corpus callosum For both corpus callosum and spinal cord injections, animals were anesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) solutions administered intraperitoneally. The assessment of complete anesthesia was confirmed by observing the hindlimb withdrawal response by pinching and compressing the animal's paw. Injection into the corpus callosum was performed using stereotaxic guidance (Plemel et al., 2018a ). Briefly, the skull was exposed, and a hole was drilled into the cranium. A 30-and-a-half-gauge needle attached to a 10µL Hamilton syringe (24575, Sigma-Aldrich) was inserted into the following coordinates: -0.8 mm anterior/posterior relative to the bregma; 0.8 mm medial/lateral to the midline; and 1.5 mm depth from the surface of the brain. A total of 1 µL of 1% LPC (62962, Sigma-Aldrich) or 0.01% LPS (L2630, Sigma-Aldrich) or a mixture of both (LPC + LPS) was injected according to the respective groups at a 0.5 µL/min rate. The Sham group was injected with the same volume of 0.9% saline. The skin of mouse was tied with suture and returned to cage. 2.3 Induction of focal demyelination in the spinal cord Mice were anesthetized as previously stated. After induction of anesthesia, a gentle incision was made in a caudal direction just below the ears, and laminectomy was done to expose the T3-T4 region of spinal cord. Then, animal was moved to stereotactic apparatus and fixed into it. Injection of toxin into the lateral white matter at T3-T4 region was performed upto 0.3mm depth adjacent to midline dorsal artery using a pulled glass capillary attached to 10µl Hamilton syringe (1725TLL, Hamilton). Injection was made according to respective groups as described before. Injecting site was tied with suture in the muscles followed by skin and kept back into the cage. 2.4 Behavioural Assessment 2.4.1 Rotarod Performance A rotarod test was performed to assess motor coordination and balance using the rotarod apparatus. In this behaviour study, mice were placed on the rotating rod with accelerations ranging from 4 to 40 rpm in 300 s. Mice were trained for 5 consecutive days. Basal readings were recorded with healthy animals before injection. After brain or spinal cord injection, rotarod performance was recorded at four-day points, i.e., Day post injection (DPI)-1, DPI-3, DPI-7, and DPI-28. Each mouse's duration on the rod before falling was measured, and the average time from three trials on the test day was calculated as the Rotarod score. To account for individual differences between mice, each mouse's Rotarod score was compared to its baseline score as a percentage for analysis. 2.4.2 Hanging wire test A hanging wire test was performed to determine muscle strength and endurance, predicting any motor deficit in limbs after injection. Basal readings were recorded with healthy animals before injection. After brain or spinal cord injection, a hanging wire test was performed at four-day points, i.e., DPI-1, DPI-3, DPI-7, and DPI-28. In this behavioural assessment, mice were placed on a cage-like wire grid and then inverted 30 cm above a padded surface. A total of three trials were performed with 15-minute intertrial intervals. Mice were allowed to hang upside down, and latency to fall was recorded (cut-off time, 300s) from an average of three trials. To account for individual differences between mice, each mouse's score was compared to its baseline score as a percentage for analysis. 2.5 Histological analysis Animals were sacrificed at four different day points after corpus callosum or spinal cord injection, i.e., at DPI-1, DPI-3, DPI-7, and DPI-28. Briefly, animals were anaesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and transcardially perfused with phosphate buffered saline (PBS), followed by 4% paraformaldehyde for fixation. Brains and spinal cords (thoracic region) were dissected and kept in 4% PFA for 24 hours for post-fixation. Samples were cryoprotected in 15% and 30% sucrose, respectively, till tissue sank into the solution, and 20 µm-thick sagittal sections (in the case of both the brain and spinal cord) were cut on a cryostat. For Nissl staining, sections were rehydrated using a series of alcohol gradients, i.e., 100%, 90%, 80%, 70%, and tap water for 5 minutes each. Sections were stained with Nissl’s solution for 10–15 minutes at 60°C and rinsed with three changes of absolute alcohol. Slides were cleared with xylene and mounted with the suitable mounting agent (DPX). For Luxol Fast Blue (LFB) staining, sections were incubated with LFB (ab150675, Abcam) for 24 hours at room temperature and rinsed in tap water. Differentiation is performed using lithium carbonate and alcohol. After rinsing in tap water, sections were counterstained with cresyl violet for 2–5 minutes. Sections were rinsed in tap water and dehydrated using three changes of absolute alcohol. Slides were cleared with xylene and mounted with the suitable mounting agent (DPX). Histological analysis was performed using bright field microscope (Leica light microscope, Germany). 2.6 Immunofluorescence Brain and spinal cord sagittal sections (20 µm thick) were washed with PBS, permeabilized by 0.3% Triton X-100 in PBS, and incubated in superblock blocking buffer (37580, Thermo Fisher Scientific) to prevent non-specific binding. Then, sections were incubated overnight at 4 ͦ C with primary antibodies against CX3CR1 (1:250, 14-6093-81, Thermo Fisher Scientific), IBA-1 (Ionized Calcium-Binding Adapter Molecule-1, 1:500, MA5-27726, Thermo Fisher Scientific), CD146 (1:500, PA5-28893, Thermo Fisher Scientific), GFAP (Glial fibrillary acidic protein, 1:1000, PA1-10004, Thermo Fisher Scientific), and MBP (Myelin basic protein, 1:2000, ab-7349, Abcam). Thoroughly washing with PBS containing 1% tween-20 sections were further incubated with secondary antibody for 1 hour at room temperature. Secondary antibodies were goat anti-rabbit Alexa Fluor 647 (1:500, ab150079, Abcam), goat anti-mouse Alexa Fluor 488 (1:500, ab150113, Abcam), goat anti-chicken Alexa Fluor 488 (1:500, ab150173, Abcam), and goat anti-rat Alexa Fluor 555 (1:500, A-21434, Thermo Fisher Scientific). After washing, sections were incubated in 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, D9542 Sigma-Aldrich) at a dilution of 1:1000 for 10 min, followed by PBS-T washing. Sections were mounted with the suitable mounting agent (DPx) and examined under confocal microscopy (Leica, Germany). 2.7 Statistical Analysis: Data are expressed as mean ± standard error of mean. Multiple groups were compared using two independent variable, Two-way ANOVA, followed by Tukey’s multiple comparisons test. P value < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism. 3. Results 3.1. Combination of lysolecithin with lipopolysaccharide impairs neuromuscular strength, enhanced neuronal loss and demyelination in corpus callosum To assess neuromuscular strength, the hanging wire test was conducted after injection with LPC, LPS and LPC + LPS. At early time points (DPI-1, 3, and 7), latency to fall increased slightly in the LPC, LPS, and LPC + LPS groups compared to the Sham group, though not significantly. However, a significant increase in latency to fall was observed at DPI-28 in the LPS and LPC + LPS groups compared to the Sham group (Fig. 1 B). Motor coordination and balance were evaluated using the rotarod test. Slight impairment was noted in all toxin groups at DPI-1, 3, and 7, but no significant differences were observed across the groups at any time point (Fig. 1 C). This indicates that while early toxin exposure did not significantly affect motor coordination, a noticeable difference in neuromuscular strength was present in the LPS and LPC + LPS groups at later time-points. To investigate structural changes in the corpus callosum, we performed LFB staining to evaluate myelin integrity and Nissl staining to assess Nissl positive glial/neuronal cells. In the Sham group, the corpus callosum was densely myelinated, with well-preserved neurons (Fig. 1 D). In contrast, complete myelin loss was observed in the LPC + LPS group at DPI-1 and 3 along with accumulation of cresyl violet positive glial cells, while the LPC and LPS groups exhibited loss in myelin but significantly lesser then combined group (Fig. 1 E). By DPI-7, the LPC and LPS groups showed significant myelin sheath degradation, whereas the LPC + LPS group showed extensive myelin loss covering a larger area and aggregation of cresyl violet positive glial/neuronal cells by DPI-28 (Supplementary Fig. 1). It signifies that combined group shows more myelin loss than individual LPC and LPS group at every time point. Nissl staining revealed a clear morphological structure in the sham group, with large Nissl + neurons and small Nissl + glial cells (Fig. 1 F). Following toxin injection, there was infiltration of small Nissl + glial cells in the corpus callosum, particularly at DPI-1 in all toxin groups. This infiltration was significantly more pronounced in the LPC and LPC + LPS groups at DPI-3, and only the LPC + LPS group showed increase in small Nissl-positive cells and loss of large Nissl + neurons at DPI-28, indicating significant persistent neuro-inflammation (Fig. 1 G). 3.2. LPC + LPS induced motor incoordination, loss of myelinated fibers and neuronal loss at chronic phase of focal demyelination in spinal cord Neuromuscular strength was also assessed after focal injection in spinal cord using the hanging wire test. A significant decrease in latency to fall was observed at DPI-1 in the LPS group compared to the Sham group. However, this effect was not sustained a later time point in any of the groups (Fig. 2 B). Motor coordination, assessed via the rotarod test, showed significant impairments in the LPC group at all-time points compared to Sham group. In the LPS group, significant impairment was observed only at DPI-7. In the LPC + LPS group, motor coordination was significantly impaired from DPI-3 to DPI-28, indicating a long-term effect on motor function (Fig. 2 C). LFB and Nissl staining were used to examine structural changes in the spinal cord. The Sham group displayed normal white matter and myelin sheath (Fig. 2 D). At DPI-1, the LPS group showed marked vacuoles at the injection site, while the LPC and LPC + LPS groups showed complete loss of myelinated fibres (Fig. 2 E). However significant myelin loss was observed on DPI-3 across all the groups. At DPI-7, the LPC group was identified by the formation of distinct vacuoles, while the LPS and LPC + LPS groups were identified by the disarray of nerve fibres. By DPI-28, the LPS groups showed some recovery with increased myelin density, but LPS group shows disarrays of nerve fibres whereas LPC + LPS group showed notable vacuole formation and continued myelin loss (supplementary Fig. 2). Nissl staining indicated a distinct demarcation between white and grey matter in the Sham group (Fig. 2 F), with fewer Nissl bodies in the ventral white and no signs of glial proliferation, chromatolysis, or neuronal apoptosis. All injection groups showed a significant increase in small Nissl + glial cells. However, the combination group (LPC + LPS) displayed the highest infiltration, blurring the distinction between white and grey matter (Fig. 2 G). 3.3 Combination of LPC + LPS compared to LPC or LPS is able to induce sustained demyelination in brain and spinal cord : We further examined the temporal dynamics of demyelination and remyelination by focussing on MBP expression. Sham group represents the highest MBP fluorescence intensity in brain and spinal cord. In brain, LPC, LPS and LPC + LPS group showed significant reduction of MBP compared to sham and extent of demyelination was not significant among all groups (Fig. 3 A- 3 B). In spinal cord, LPC, LPS and especially LPC + LPS groups showed significant MBP reduction at DPI-1, 3, and 7. At DPI-28 LPC + LPS showed highest reduction even significant than LPC (Fig. 3 C- 3 D). 3.4 Pronounced activation of astrocyte and endothelial cells in combination of LPC + LPS toxins Astrogliosis, indicated by the presence of reactive astrocytes, were assessed in relation to blood vessel integrity, which is critical for maintaining the BBB/BSCB. To investigate this, we examined the expression of astrocytes and blood vessels in the brain and spinal cord following focal demyelination induced by LPC, LPS, and a combination of LPC and LPS (Fig. 4 A- 4 C). In brain, the expression of GFAP is dramatically high compared to sham, LPC and LPS groups signifies the activated state of astroglia. Next the expression of CD146 was highly upregulated at day-1 in LPC + LPS group compared to the sham, LPC and LPC + LPS groups followed by moderate increase. This signifies the endothelial activation preceds the astrocytic activation in combination group. In spinal cord, we have found the increased expression of GFAP over time, especially in LPC + LPS group significantly at DPI-28. Next, we have found upregulated expression of CD146 in LPS and LPC + LPS groups but not in alone LPC group. Moreover, in LPC + LPS the expression was significantly high compared to LPC alone (Fig. 4 D- 4 F). This suggests the significant activation of astrocytic and endothelial cells. 3.5 LPC + LPS induced neuroinflammatory chemokine on reactive microglia at later phase of focal demyelination C-X-C motif chemokine receptor 1 (CX3CR1), a chemokine receptor on microglia, was assessed as a marker of neuroinflammation. In brain, Iba-1 expression is markedly increased in the LPC + LPS group at DPI-3, indicating the robust microglial activation. LPC and LPS showed mild increase in Iba-1 expression. CX3CR1 expression is significantly upregulated in LPC + LPS group at DPI-7 and normalised upto DPI-28. LPC and LPS groups alone showed moderate increase in CX3CR1 expression (Fig. 5 A- 5 C). In spinal cord, the Iba-1 expression was markedly increased in LPC + LPS group, peaking at DPI-3. Microglia were found to be upregulated in LPC and LPS groups alone but not significantly. CX3CR1 expression is significantly upregulated in all groups but highly expressed at DPI-3 and 28. LPC and LPS groups also showed the dynamic and time dependent significant upregulated expression. This suggests that rapid microglia activation and proliferation in response to demyelination and inflammation. Sustained increased expression of CX3CR1 suggested the recruitment of microglia towards the chronic inflammatory state (Fig. 5 D- 5 F) 4. Discussion Myelination is essential for the health of neurons, as the myelin sheath facilitates rapid signal conduction along axons and maintains structural integrity. Damage to the myelin sheath leads to demyelination, often due to immune system attacks and neuroinflammation (Abbott et al., 2010 ). Numerous studies have investigated demyelination in various neuroinflammatory diseases, such as multiple sclerosis, traumatic injuries, and neurodegeneration (Haider et al., 2016 ; Kulkarni et al., 2022 ; Zirngibl et al., 2022 ). However, replicating the complex pathology of demyelination in animal models has been challenging. Existing toxin models, such as LPC, LPS and ethidium bromide for focal and diffuse demyelination, and cuprizone and EAE for global demyelination, do not fully capture the complexity of demyelination (Lassmann and Bradl, 2017 ; Gharagozloo et al., 2022 ). To address this gap, we developed a model using a combination of LPC and LPS injections into the corpus callosum and the thoracic region of the spinal cord. LPC disrupts the cell membrane, increasing its permeability and allowing chemokines to infiltrate, causing localized myelin damage. LPS, through toll-like receptor activation, elicits a strong immune response. In our study, the combined effect of LPC and LPS injections in the corpus callosum and spinal cord led to neuromuscular strength impairment and motor coordination deficits. Histological analyses with LFB revealed disorganized nerve fibers, vacuole formation. Nissl staining revealed infiltration of small nissl + bodies. While remyelination typically follows demyelination in toxin models, our findings indicated a delayed remyelination phase in the LPC + LPS group, suggesting persistent demyelination in both the brain and spinal cord. Inflammatory conditions associated with demyelination can significantly impact the integrity of the blood-brain barrier (BBB) (Kirk et al., 2003 ; Zierfuss et al., 2024 ), blood-spinal cord barrier (BSCB) (Aubé et al., 2014 ), and modulates glial cells activity (McQuaid et al., 2009 ). In mammals and most vertebrates, BBB/BSCB is formed by endothelial cells connected by tight junctions, providing high electrical resistance and safeguarding neurons (Abbott et al., 2010 ; Bartanusz et al., 2011 ). Disruption of endothelial cells can damage the BBB/BSCB, enhancing the trafficking of immune cells and neuroinflammation, resulting in the degradation of the myelin sheath and demyelination (Berghoff et al., 2017 ; Goyal and Kumar, 2023 ). Studies have shown that brain endothelial cells increase the proliferation of oligodendrocyte precursor cells, which are the precursors to mature oligodendrocytes involved in myelination (Arai and Lo, 2009 ). During the stages of BBB induction and maturation, melanoma cell adhesion molecules, growth factors, and their receptors exhibit dynamic expression patterns in the cerebrovascular, coordinating interactions between endothelial cells and pericytes and orchestrating the spatiotemporal development of the BBB (Chen et al., 2017 ; Raj et al., 2024 ). In neuroinflammatory conditions such as multiple sclerosis (MS), CD146 gets upregulated on BBB endothelial cells, promoting the transmigration of inflammatory cells into the CNS due to BBB dysfunction (Arai and Lo, 2009 ). Prior studies have shown that BBB/BSCB hyperpermeability and vascular dysfunction are significant contributors to multiple sclerosis (Berghoff et al., 2017 ; Cashion et al., 2023 ). Our data demonstrated a disrupted vascular integrity and marked astrogliosis in the spinal cord and brain, particularly in the LPC + LPS group. These data suggests that the unique environment of the BSCB, which is more permeable than the BBB, may account for the differential impact observed between the spinal cord and brain. In case of CC demyelination endothelial dysfunction precedes the astrogliosis suggesting the causal factor for delayed remyelination. Subsequently in spinal cord demyelination, our data suggest that astrogliosis is pronounced in later phase of demyelination in LPC + LPS and LPC group, however the endothelial dysfunction was dynamic and significantly high than other groups. The differential expression of vascular dysfunction and astrogliosis in the brain and spinal cord can be explained by the unique environment of the BBB and BSCB. A report highlighted that BSCB is highly permeable to a variety of substances, such as TNF-α, inulin, and mannitol (Daniel et al., 1985 ; Pan et al., 1997 ). Another report highlighted that spinal cord pericyte coverage is less than brain pericyte coverage, thus contributing to higher permeability. Along with this, tight junction proteins such as zona occludens-1 and occludin are also less common than in the brain (Winkler et al., 2012 ). Thus, in our study, it is possible that the combination group of toxins has a greater impact on vascular integrity, hyperpermeability, and astrogliosis. Chronic activation of glial cells, such as microglia and astrocytes, is also observed in MS. These cells release various pro-inflammatory cytokines and chemokines, including TNF-α, IL-1β, CCL2, CCL5, and CXCL12. The binding of these molecules to their respective receptors aggravates the process of demyelination (Ponath et al., 2018 ; Voet et al., 2019 ; Healy et al., 2022 ). A study suggested that resident microglia are crucial for the maintenance of myelin integrity (McNamara et al., 2023 ). In contrast, another study highlighted that microglia adapt the phagocytosis-enhanced phenotype during demyelinated lesions and play a central role in neuroinflammation (Slobodov et al., 2001 ). Consistent with this, previous studies demonstrated that the dynamic response of microglia aggravates demyelination (Chu et al., 2019 ). Moreover, the lesion-associated microglia are more prominent than the infiltrating macrophages in the CNS following the LPC model of demyelination (Plemel et al., 2020 ). Another study suggested that microglia markedly increased the numbers and expanded by releasing cytokine colony stimulating factor during the demyelination following the cuprizone model. However, the number decreased after pre-depleting microglia with cytokine inhibitors (Marzan et al., 2021 ). Neuron-derived chemokine fractalkine binds to the CX3CR1 receptor on microglia. However, the defective signalling of CX3CR1 and its ligand fractalkine aggravates neuroinflammation and demyelination (Mendiola et al., 2022 ). A previous study highlighted that mRNA expression of CX3CR1 increases at later time points after SCI and mice deficient CX3CR1 gene recovered from SCI compared to the wild-type mice (Donnelly et al., 2011 ). Our data indicated that Iba-1 expression was highly expressed at acute phase of DPI-3, more pronounced in LPC + LPS group. The expression of CX3CR1 was sustained and expressed throughout in case of spinal cord promoting sustained demyelination. These results suggests that LPC + LPS exacerbates the vascular and glial pathology, highlighting the importance of vascular-glial interactions in demyelinating diseases. Despite the novelty of these findings in the LPC + LPS group, there are certain limitations that need to be addressed in future research. Further, we need to explore the downstream pathways to establish the synergistic effect of the combination group on demyelination. Secondly, we need to see the effect of combined toxins in oligodendrocyte development and myelination. Next, we have to provide some major evidence that the combination group has a greater impact on the spinal cord microenvironment than the brain. 5. Conclusion Our study provides a comprehensive understanding of the mechanisms underlying persistent demyelination. The synergistic effects of LPC and LPS induced a cascade of events, including motor dysfunction, neuronal loss, persistent demyelination, and neuroinflammation characterized by astrogliosis, endothelial dysfunction, and microglial activation. This novel model offers a valuable platform for studying the complex interplay between myelin damage and immune activation and their contribution to persistent demyelination. By identifying these key pathological processes, our findings may facilitate the development of innovative therapeutic strategies aimed at promoting remyelination and mitigating neuroinflammation. Declarations Author Contributions DG: Investigation, Data curation, Writing and editing the draft. KD: Investigation, Data curation and project administration. SR: Investigation, Data curation and project administration. PM: Investigation and project administration. RPN: Analysis and editing of the draft. NS: Literature, designing and editing of the manuscript. HK: Concepts and design of study, supervision, visualization, review and editing of the final draft. Acknowledgment We thank the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India and NIPER Ahmedabad. Funding Declaration The current work was supported by the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India and NIPER Ahmedabad. 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Healy LM, Stratton JA, Kuhlmann T, Antel J (2022) The role of glial cells in multiple sclerosis disease progression. Nature Reviews Neurology 18:237-248. Hollis ER, 2nd, Ishiko N, Tolentino K, Doherty E, Rodriguez MJ, Calcutt NA, Zou Y (2015) A novel and robust conditioning lesion induced by ethidium bromide. Exp Neurol 265:30-39. Kirk J, Plumb J, Mirakhur M, McQuaid S (2003) Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood–brain barrier leakage and active demyelination. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland 201:319-327. Kulkarni R, Thakur A, Kumar H (2022) Microtubule Dynamics Following Central and Peripheral Nervous System Axotomy. ACS Chemical Neuroscience 13:1358-1369. Lassmann H, Bradl M (2017) Multiple sclerosis: experimental models and reality. Acta neuropathologica 133:223-244. Marzan DE, Brügger‐Verdon V, West BL, Liddelow S, Samanta J, Salzer JL (2021) Activated microglia drive demyelination via CSF1R signaling. Glia 69:1583-1604. McNamara NB et al. (2023) Microglia regulate central nervous system myelin growth and integrity. Nature 613:120-129. McQuaid S, Cunnea P, McMahon J, Fitzgerald U (2009) The effects of blood–brain barrier disruption on glial cell function in multiple sclerosis. Biochemical Society Transactions 37:329-331. Melamed E, Palmer JL, Fonken C (2022) Advantages and limitations of experimental autoimmune encephalomyelitis in breaking down the role of the gut microbiome in multiple sclerosis. Front Mol Neurosci 15:1019877. Mendiola AS, Church KA, Cardona SM, Vanegas D, Garcia SA, Macklin W, Lira SA, Ransohoff RM, Kokovay E, Lin CHA (2022) Defective fractalkine‐CX3CR1 signaling aggravates neuroinflammation and affects recovery from cuprizone‐induced demyelination. Journal of neurochemistry 162:430-443. Pan W, Banks WA, Kastin AJ (1997) Blood–brain barrier permeability to ebiratide and TNF in acute spinal cord injury. Experimental neurology 146:367-373. Plemel JR, Michaels NJ, Weishaupt N, Caprariello AV, Keough MB, Rogers JA, Yukseloglu A, Lim J, Patel VV, Rawji KS (2018a) Mechanisms of lysophosphatidylcholine‐induced demyelination: A primary lipid disrupting myelinopathy. Glia 66:327-347. Plemel JR, Michaels NJ, Weishaupt N, Caprariello AV, Keough MB, Rogers JA, Yukseloglu A, Lim J, Patel VV, Rawji KS, Jensen SK, Teo W, Heyne B, Whitehead SN, Stys PK, Yong VW (2018b) Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy. Glia 66:327-347. Plemel JR et al. (2020) Microglia response following acute demyelination is heterogeneous and limits infiltrating macrophage dispersion. Science Advances 6:eaay6324. Ponath G, Park C, Pitt D (2018) The role of astrocytes in multiple sclerosis. Frontiers in immunology 9:217. Popescu BFG, Lucchinetti CF (2012) Pathology of demyelinating diseases. Annual Review of Pathology: Mechanisms of Disease 7:185-217. Raj S, Sarangi P, Goyal D, Kumar H (2024) The Hidden Hand in White Matter: Pericytes and the Puzzle of Demyelination. ACS Pharmacology & Translational Science. Sherman DL, Brophy PJ (2005) Mechanisms of axon ensheathment and myelin growth. Nature Reviews Neuroscience 6:683-690. Shi H, Hu X, Leak RK, Shi Y, An C, Suenaga J, Chen J, Gao Y (2015) Demyelination as a rational therapeutic target for ischemic or traumatic brain injury. Experimental neurology 272:17-25. Slobodov U, Reichert F, Mirski R, Rotshenker S (2001) Distinct Inflammatory Stimuli Induce Different Patterns of Myelin Phagocytosis and Degradation in Recruited Macrophages. Experimental Neurology 167:401-409. Torkildsen O, Brunborg LA, Myhr KM, Bø L (2008) The cuprizone model for demyelination. Acta Neurol Scand Suppl 188:72-76. Voet S, Prinz M, van Loo G (2019) Microglia in central nervous system inflammation and multiple sclerosis pathology. Trends in molecular medicine 25:112-123. Winkler EA, Sengillo JD, Bell RD, Wang J, Zlokovic BV (2012) Blood–Spinal Cord Barrier Pericyte Reductions Contribute to Increased Capillary Permeability. Journal of Cerebral Blood Flow & Metabolism 32:1841-1852. Zierfuss B, Larochelle C, Prat A (2024) Blood–brain barrier dysfunction in multiple sclerosis: Causes, consequences, and potential effects of therapies. The Lancet Neurology 23:95-109. Zirngibl M, Assinck P, Sizov A, Caprariello AV, Plemel JR (2022) Oligodendrocyte death and myelin loss in the cuprizone model: an updated overview of the intrinsic and extrinsic causes of cuprizone demyelination. Molecular Neurodegeneration 17:34. Additional Declarations No competing interests reported. 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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-8517400","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592097847,"identity":"84dd31ad-a1ce-4307-9174-68a3a32ffc5f","order_by":0,"name":"Divya Goyal","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Divya","middleName":"","lastName":"Goyal","suffix":""},{"id":592097848,"identity":"d30e7b01-0743-4646-9e9c-9b3cba7286fc","order_by":1,"name":"Krutika H. Dobariya","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Krutika","middleName":"H.","lastName":"Dobariya","suffix":""},{"id":592097849,"identity":"d79726db-4eee-4a9e-9b3e-44d2ed426dbb","order_by":2,"name":"Siddharth Raj","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Siddharth","middleName":"","lastName":"Raj","suffix":""},{"id":592097850,"identity":"4de890b4-18a5-4a7e-8401-d4ba19b67aef","order_by":3,"name":"Piyush Maheshkar","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Piyush","middleName":"","lastName":"Maheshkar","suffix":""},{"id":592097851,"identity":"6789e429-63ee-4e7b-8a4c-cf40ec66f3ee","order_by":4,"name":"Rudra Prasad Naik","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Rudra","middleName":"Prasad","lastName":"Naik","suffix":""},{"id":592097852,"identity":"aeb61fab-fbb3-4253-b7a8-7b06f8aaa69a","order_by":5,"name":"Nidhi Singh","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Nidhi","middleName":"","lastName":"Singh","suffix":""},{"id":592097854,"identity":"46f7dccc-c788-493b-a269-fb1de5ea4d4f","order_by":6,"name":"Hemant Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACAwhlw8AGohgboMIJBLUkpJGu5TCEB9eCD5izdyd+LvxxPo9PuvnYh587tjGYt7c/YHi4A7cWy56zm6VnJNwuZpM5ljyz98xtBpkzZwwYEs/gcdiN3A3SPAm3E9skcowZeNtuM0hI5DAwJLbh0XL/7ebfPAnngFryPzP+BWmRf/4Av5YbvNuAthwA2cLMDLEFGCZ4tZzJ3WbNk5ac2CZzzJhZtu02jwRPjsEBvFqOn918m8fGLnH+7ObHjG/bbstJsB9/+PAnHi0IIAGheEDEAWI0wLWMglEwCkbBKMAAAETJUX3pdBbQAAAAAElFTkSuQmCC","orcid":"","institution":"National Institute of Pharmaceutical Education and Research","correspondingAuthor":true,"prefix":"","firstName":"Hemant","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2026-01-05 06:08:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8517400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8517400/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102858463,"identity":"56607deb-398f-442f-9cdb-e6b7dec8379d","added_by":"auto","created_at":"2026-02-17 15:44:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2066588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of\u003c/strong\u003e \u003cstrong\u003eLysolecithin and Lipopolysaccharide (LPC+LPS) induced neuromuscular strength impairment, neuronal loss and loss of myelinated fibers at chronic phase of focal demyelination in corpus callosum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representing the illustration of focal injection at corpus callosum in brain, (b) Hanging wire score (normalized to each baseline) at different time-points. For each time-point, statistical significance was noted only in LPC+LPS when compared to the Sham group (**p\u0026lt; 0.01) at dpi-28. For each group, no statistical significance between different time-points except when compared to basal ($p\u0026lt;0.05, $$$p\u0026lt;0.001) and dpi-1 (##p\u0026lt;0.01) in LPC+LPS and LPS group. (n=6-12). (c) Rotarod performance (normalized to each baseline) at different time-points. No statistical significance between the LPC or LPS or LPC+LPS group and Sham group were observed at any given time-point; for each group, no statistical significance between different time-points was found. (n=6-12). (d) representative image of Luxol fast blue staining of Sham group (Black box demonstrates location of magnified regions), (e) representative images of Luxol fast blue staining of LPC, LPS and LPC+LPS groups at various day points, (f) representative image of Nissl statining of Sham group (Black box demonstrates location of magnified regions), (g) representative images of Nissl staining of LPC, LPS and LPC+LPS groups at various day points (scale bar, 500\u003cem\u003eµ\u003c/em\u003em) Arrowheads indicating the corpus callosum.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/20b2fcf5f021759e0fa1680f.png"},{"id":102858465,"identity":"16856f91-2acc-40fb-8505-0d53a2ffcd6e","added_by":"auto","created_at":"2026-02-17 15:44:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2032850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of LPC and LPS induced motor incoordination, loss of myelinated fibers and neuronal loss at chronic phase of focal demyelination in spinal cord\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representing the illustration of focal injection at ventarl white matter of spinal cord, (b) Hanging wire score (normalized to each baseline) at different time-points. For each time-point, handing wire score was compared Sham group (*p\u0026lt;0.05) and LPC group (%p\u0026lt;0.05) ;For each group, no statistical significance between different time-points was found. (n=6-12) (c) Rotarod performance (normalized to each baseline) at different time-points. For each time-point, rotarod score was compared Sham group (*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001) and LPS group (\u0026amp;p\u0026lt;0.05); For each group, no statistical significance between different time-points except when compared to basal ($p\u0026lt;0.05, $$p\u0026lt;0.01, $$$p\u0026lt;0.001, $$$$p\u0026lt;0.0001) and dpi-1 (#p\u0026lt;0.05, ##p\u0026lt;0.01). (n=6-12), (d) representative image of Luxol fast blue staining in Sham group of spinal cord (Black box demonstrates location of magnified regions), (e) representative images of Luxol fast blue staining of LPC, LPS and LPC+LPS groups at different stages in spinal cord, (f) representative image of Nissl statining in Sham group of spinal cord (Black box demonstrates location of magnified regions), (g) representative images of Nissl staining of LPC, LPS and LPC+LPS groups at different stages showing different patterns of neurodegeneration in spinal cord (scale bar, 200\u003cem\u003eµ\u003c/em\u003em).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/57b7a83df8b8f1f80e1015fc.png"},{"id":102858468,"identity":"e2bf3735-d4c8-4d3e-b71c-0da2a89bc935","added_by":"auto","created_at":"2026-02-17 15:44:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3046489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombination of LPC+LPS but not alone LPC or LPS is able to induce delayed remyelination. \u003c/strong\u003e(A) representative images of immunohistochemistry showing the levels of Myelin basic protein (MBP) expression in brain marking the early demyelination followed by remyelination in LPC and LPS group and sustained demyelination in LPC+LPS group. (3B) represents the graph of quantification of MBP expression in brain. 3(c) representative images of immunohistochemistry showing the levels of MBP expression in spinal cord signifying the early demyelination in LPC and LPS group followed by remyelination at later stage in LPC group and amplication of MBP expression in LPS group at later stages, whereas, denoting sustained demyelination in LPC+LPS group at later stages. 3(d) represents the graph of quantification for MBP expression in spinal cord. Yellow box demonstrates location of magnified regions in IHC (scale bar, 25\u003cem\u003eµ\u003c/em\u003em).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eFor each time-point (n=3), each toxin group was compared to the Sham group (*𝑝\u0026lt;0.05), (∗∗𝑝\u0026lt;0.01), (***𝑝\u0026lt;0.001) and to the LPC group (#𝑝\u0026lt;0.05), (##𝑝\u0026lt;0.05), (###𝑝\u0026lt;0.01), and LPS group (@𝑝\u0026lt;0.05), (@@𝑝\u0026lt;0.01), (@@@𝑝\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/4874749782426789a935e274.png"},{"id":102858467,"identity":"33e2f0a7-28d7-4647-b56b-6245563ed795","added_by":"auto","created_at":"2026-02-17 15:44:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1516669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrogliosis and blood vessel dysfunction are highly associated in LPC+LPS Induced focal demyelination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e4 (A) Representative immunofluorescence images showing Co-localization of Glial fibrillary acidic proteins (GFAP) -positive astrocytes (green) and CD146-positive endothelial cells (red), in the brain following induction of focal demyelination. 4(B) represents the graph of quantification for expression of GFAP in the brain whereas 4(C) represents the graph of quantification for CD-146 in the brain. 4(D) Represents immunofluorescence images showing Co-localization of GFAP-positive astrocytes (green) and CD146-positive endothelial cells (red), in the Spinal cord following induction of focal demyelination. 4(E) represents the graph of quantification for expression of GFAP in the spinal cord and 4(F) represents the graph for quantification of CD-146 expression in the spinal cord. (scale bar, 25\u003cem\u003eµ\u003c/em\u003em).\u003c/p\u003e\n\u003cp\u003eFor each time-point (n=3), each toxin group was compared to the Sham group (*𝑝\u0026lt;0.05), (∗∗𝑝\u0026lt;0.01), (***𝑝\u0026lt;0.001) and to the LPC group (#𝑝\u0026lt;0.05), (##𝑝\u0026lt;0.05), (###𝑝\u0026lt;0.01), and LPS group (@𝑝\u0026lt;0.05), (@@𝑝\u0026lt;0.01), (@@@𝑝\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/7a59f481d40f568b2553c0f8.png"},{"id":102963820,"identity":"5e0fbcee-3e57-4131-91b8-60eeb332b4c0","added_by":"auto","created_at":"2026-02-19 04:20:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3962946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPC+LPS induced neuroinflammatory chemokine on reactive microglia at later phase of focal demyelination \u003c/strong\u003e5(A)\u003cstrong\u003e \u003c/strong\u003eRepresentative immunofluorescence images showing C-X-C motif chemokine receptor 1 (CX3CR1) (red) and Ionized calcium-binding adapter molecule-1 (Iba-1) (green) expression in the brain at DPI-1, DPI-3, DPI-7, and DPI-28 after LPC, LPS, and LPC+LPS treatments. 5\u003cstrong\u003e(\u003c/strong\u003eB\u003cstrong\u003e) \u003c/strong\u003erepresents the\u003cstrong\u003e \u003c/strong\u003egraph of quantification for the expression of CX3CR1 expression in the brain whereas 5(C) represents the graph of quantification for the expression of Iba-1 in the brain. 5(D) Representative immunofluorescence images showing CX3CR1 (red) and Iba-1 (green) expression in the spinal cord at DPI-1, DPI-3, DPI-7, and DPI-28 after LPC, LPS, and LPC+LPS treatments. 5(E) represents the graph of quantification for the expression of CX3CR1 in the spinal cord whereas 5(F) represents the graph of quantification of the expression of the Iba-1 in the spinal cord\u003c/p\u003e\n\u003cp\u003e(scale bar, 25\u003cem\u003eµ\u003c/em\u003em).\u003c/p\u003e\n\u003cp\u003eFor each time-point (n=3), each toxin group was compared to the Sham group (*𝑝\u0026lt;0.05), (∗∗𝑝\u0026lt;0.01), (***𝑝\u0026lt;0.001) and to the LPC group (#𝑝\u0026lt;0.05), (##𝑝\u0026lt;0.05), (###𝑝\u0026lt;0.01), and LPS group (@𝑝\u0026lt;0.05), (@@𝑝\u0026lt;0.01), (@@@𝑝\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/1ddc6c1897cd4b1cef48a05b.png"},{"id":105563287,"identity":"0531abb1-0b95-4ed9-9405-d20ff4102e44","added_by":"auto","created_at":"2026-03-27 12:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12352609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/73ee4f8d-1ad4-45c8-9278-5b4c571867f7.pdf"},{"id":102963116,"identity":"b7209836-9a08-458c-9240-6ba716b14a9d","added_by":"auto","created_at":"2026-02-19 04:13:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":54217,"visible":true,"origin":"","legend":"","description":"","filename":"SupplymentaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-8517400/v1/3c54b6e66684ee70d4d1fe38.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regional Vulnerability to Demyelination: A Comparative Study of the Effects of LPC, LPS, and Combined Toxins in Corpus Callosum and Spinal Cord","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn the central nervous system (CNS), myelin is a lipid-rich protective sheath that wraps around axons, facilitating communication between neurons by enabling rapid impulse propagation (Bercury and Macklin, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The breakdown or loss of myelin leads to demyelination, a process associated with various neurodegenerative and pathological conditions, including multiple sclerosis (MS), Parkinson\u0026rsquo;s disease, Alzheimer\u0026rsquo;s disease, acute disseminated encephalomyelitis and traumatic injuries of the brain and spinal cord (Shi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dong et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Myelin constitutes the white matter within brain regions and surrounds the spinal cord periphery. When myelin is disrupted, it results in white matter loss, neuroinflammation, and deficits in motor behaviour (Sherman and Brophy, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Popescu and Lucchinetti, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral \u003cem\u003ein vivo\u003c/em\u003e models of demyelination have been established to understand this process. The most widely used is the experimental autoimmune encephalomyelitis (EAE) model, which is induced through immunization with antigens (Constantinescu et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Glatigny and Bettelli, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), mimicking the autoimmune nature of MS, although it lacks the genetic and environmental factors contributing to demyelination (Melamed et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The cuprizone model, a copper chelator, induces demyelination by impairing oligodendrocyte function (Torkildsen et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zirngibl et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), though it causes non-specific demyelination across various brain regions and is typically transient (Zirngibl et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Alternatively, toxins-induced models, such as those using lysolecithin (LPC) and ethidium bromide, directly damage myelin without immune sensitization (Hollis et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Plemel et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e), primarily focusing on primary demyelination and targets myelinating cells. While such models lack the immune-mediated complexity of diseases like MS, they allow for a more controlled investigation of demyelination processes.\u003c/p\u003e \u003cp\u003eLPC, integrates into the cellular membrane to increase permeability and disrupts myelin integrity, though it is short-lived and is cleared from white matter within 24 hours (Plemel et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). Conversely, LPS, a potent inflammatory mediator, binds to TLR4 receptors on microglia, triggering their activation and promoting the release of pro-inflammatory cytokines and chemokines, which drive neuroinflammation (Felts et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe distinct mechanisms of LPC and LPS led us to explore a novel combined model. We hypothesized that the synergistic effects of these two toxins would produce a more consistent and robust demyelination and neuroinflammatory phenotype, potentially enhancing neurovascular and glial responses. By combining LPS and LPC, our goal is to establish a more comprehensive and clinically relevant model for studying demyelinating diseases.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eAdult C57/BL6 male mice (Zydus, Ahmedabad, India), were used to carry out the experimental study. The mice, aged 6\u0026ndash;8 weeks and weighing around 25\u0026ndash;30 g, were randomly assigned into four groups: Sham, LPC, LPS, and LPC\u0026thinsp;+\u0026thinsp;LPS. Each group consisted of mice allocated for injection studies either in the spinal cord or corpus callosum. All animals were housed in a facility with controlled environmental conditions, maintaining humidity at 55\u0026ndash;65%, temperature at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C, and a 12-hour light-dark cycle. Food and water were provided ad libitum. All procedures and experiments were conducted in accordance with the protocol (IAEC/2023/032) approved by the Institutional Animal Ethics Committee of NIPER-Ahmedabad and the principles of laboratory animal care.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Induction of focal demyelination in corpus callosum\u003c/h2\u003e \u003cp\u003eFor both corpus callosum and spinal cord injections, animals were anesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) solutions administered intraperitoneally. The assessment of complete anesthesia was confirmed by observing the hindlimb withdrawal response by pinching and compressing the animal's paw. Injection into the corpus callosum was performed using stereotaxic guidance (Plemel et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e). Briefly, the skull was exposed, and a hole was drilled into the cranium. A 30-and-a-half-gauge needle attached to a 10\u0026micro;L Hamilton syringe (24575, Sigma-Aldrich) was inserted into the following coordinates: -0.8 mm anterior/posterior relative to the bregma; 0.8 mm medial/lateral to the midline; and 1.5 mm depth from the surface of the brain. A total of 1 \u0026micro;L of 1% LPC (62962, Sigma-Aldrich) or 0.01% LPS (L2630, Sigma-Aldrich) or a mixture of both (LPC\u0026thinsp;+\u0026thinsp;LPS) was injected according to the respective groups at a 0.5 \u0026micro;L/min rate. The Sham group was injected with the same volume of 0.9% saline. The skin of mouse was tied with suture and returned to cage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Induction of focal demyelination in the spinal cord\u003c/h2\u003e \u003cp\u003eMice were anesthetized as previously stated. After induction of anesthesia, a gentle incision was made in a caudal direction just below the ears, and laminectomy was done to expose the T3-T4 region of spinal cord. Then, animal was moved to stereotactic apparatus and fixed into it. Injection of toxin into the lateral white matter at T3-T4 region was performed upto 0.3mm depth adjacent to midline dorsal artery using a pulled glass capillary attached to 10\u0026micro;l Hamilton syringe (1725TLL, Hamilton). Injection was made according to respective groups as described before. Injecting site was tied with suture in the muscles followed by skin and kept back into the cage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Behavioural Assessment\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Rotarod Performance\u003c/h2\u003e \u003cp\u003eA rotarod test was performed to assess motor coordination and balance using the rotarod apparatus. In this behaviour study, mice were placed on the rotating rod with accelerations ranging from 4 to 40 rpm in 300 s. Mice were trained for 5 consecutive days. Basal readings were recorded with healthy animals before injection. After brain or spinal cord injection, rotarod performance was recorded at four-day points, i.e., Day post injection (DPI)-1, DPI-3, DPI-7, and DPI-28. Each mouse's duration on the rod before falling was measured, and the average time from three trials on the test day was calculated as the Rotarod score. To account for individual differences between mice, each mouse's Rotarod score was compared to its baseline score as a percentage for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Hanging wire test\u003c/h2\u003e \u003cp\u003eA hanging wire test was performed to determine muscle strength and endurance, predicting any motor deficit in limbs after injection. Basal readings were recorded with healthy animals before injection. After brain or spinal cord injection, a hanging wire test was performed at four-day points, i.e., DPI-1, DPI-3, DPI-7, and DPI-28. In this behavioural assessment, mice were placed on a cage-like wire grid and then inverted 30 cm above a padded surface. A total of three trials were performed with 15-minute intertrial intervals. Mice were allowed to hang upside down, and latency to fall was recorded (cut-off time, 300s) from an average of three trials. To account for individual differences between mice, each mouse's score was compared to its baseline score as a percentage for analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histological analysis\u003c/h2\u003e \u003cp\u003eAnimals were sacrificed at four different day points after corpus callosum or spinal cord injection, i.e., at DPI-1, DPI-3, DPI-7, and DPI-28. Briefly, animals were anaesthetized with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and transcardially perfused with phosphate buffered saline (PBS), followed by 4% paraformaldehyde for fixation. Brains and spinal cords (thoracic region) were dissected and kept in 4% PFA for 24 hours for post-fixation. Samples were cryoprotected in 15% and 30% sucrose, respectively, till tissue sank into the solution, and 20 \u0026micro;m-thick sagittal sections (in the case of both the brain and spinal cord) were cut on a cryostat. For Nissl staining, sections were rehydrated using a series of alcohol gradients, i.e., 100%, 90%, 80%, 70%, and tap water for 5 minutes each. Sections were stained with Nissl\u0026rsquo;s solution for 10\u0026ndash;15 minutes at 60\u0026deg;C and rinsed with three changes of absolute alcohol. Slides were cleared with xylene and mounted with the suitable mounting agent (DPX). For Luxol Fast Blue (LFB) staining, sections were incubated with LFB (ab150675, Abcam) for 24 hours at room temperature and rinsed in tap water. Differentiation is performed using lithium carbonate and alcohol. After rinsing in tap water, sections were counterstained with cresyl violet for 2\u0026ndash;5 minutes. Sections were rinsed in tap water and dehydrated using three changes of absolute alcohol. Slides were cleared with xylene and mounted with the suitable mounting agent (DPX). Histological analysis was performed using bright field microscope (Leica light microscope, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunofluorescence\u003c/h2\u003e \u003cp\u003eBrain and spinal cord sagittal sections (20 \u0026micro;m thick) were washed with PBS, permeabilized by 0.3% Triton X-100 in PBS, and incubated in superblock blocking buffer (37580, Thermo Fisher Scientific) to prevent non-specific binding. Then, sections were incubated overnight at 4 ͦ C with primary antibodies against CX3CR1 (1:250, 14-6093-81, Thermo Fisher Scientific), IBA-1 (Ionized Calcium-Binding Adapter Molecule-1, 1:500, MA5-27726, Thermo Fisher Scientific), CD146 (1:500, PA5-28893, Thermo Fisher Scientific), GFAP (Glial fibrillary acidic protein, 1:1000, PA1-10004, Thermo Fisher Scientific), and MBP (Myelin basic protein, 1:2000, ab-7349, Abcam). Thoroughly washing with PBS containing 1% tween-20 sections were further incubated with secondary antibody for 1 hour at room temperature. Secondary antibodies were goat anti-rabbit Alexa Fluor 647 (1:500, ab150079, Abcam), goat anti-mouse Alexa Fluor 488 (1:500, ab150113, Abcam), goat anti-chicken Alexa Fluor 488 (1:500, ab150173, Abcam), and goat anti-rat Alexa Fluor 555 (1:500, A-21434, Thermo Fisher Scientific). After washing, sections were incubated in 4\u0026prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI, D9542 Sigma-Aldrich) at a dilution of 1:1000 for 10 min, followed by PBS-T washing. Sections were mounted with the suitable mounting agent (DPx) and examined under confocal microscopy (Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis:\u003c/h2\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean. Multiple groups were compared using two independent variable, Two-way ANOVA, followed by Tukey\u0026rsquo;s multiple comparisons test. P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1. Combination of lysolecithin with lipopolysaccharide impairs neuromuscular strength, enhanced neuronal loss and demyelination in corpus callosum\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess neuromuscular strength, the hanging wire test was conducted after injection with LPC, LPS and LPC\u0026thinsp;+\u0026thinsp;LPS. At early time points (DPI-1, 3, and 7), latency to fall increased slightly in the LPC, LPS, and LPC\u0026thinsp;+\u0026thinsp;LPS groups compared to the Sham group, though not significantly. However, a significant increase in latency to fall was observed at DPI-28 in the LPS and LPC\u0026thinsp;+\u0026thinsp;LPS groups compared to the Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Motor coordination and balance were evaluated using the rotarod test. Slight impairment was noted in all toxin groups at DPI-1, 3, and 7, but no significant differences were observed across the groups at any time point (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This indicates that while early toxin exposure did not significantly affect motor coordination, a noticeable difference in neuromuscular strength was present in the LPS and LPC\u0026thinsp;+\u0026thinsp;LPS groups at later time-points.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate structural changes in the corpus callosum, we performed LFB staining to evaluate myelin integrity and Nissl staining to assess Nissl positive glial/neuronal cells. In the Sham group, the corpus callosum was densely myelinated, with well-preserved neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). In contrast, complete myelin loss was observed in the LPC\u0026thinsp;+\u0026thinsp;LPS group at DPI-1 and 3 along with accumulation of cresyl violet positive glial cells, while the LPC and LPS groups exhibited loss in myelin but significantly lesser then combined group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). By DPI-7, the LPC and LPS groups showed significant myelin sheath degradation, whereas the LPC\u0026thinsp;+\u0026thinsp;LPS group showed extensive myelin loss covering a larger area and aggregation of cresyl violet positive glial/neuronal cells by DPI-28 (Supplementary Fig.\u0026nbsp;1). It signifies that combined group shows more myelin loss than individual LPC and LPS group at every time point. Nissl staining revealed a clear morphological structure in the sham group, with large Nissl\u003csup\u003e+\u003c/sup\u003e neurons and small Nissl\u003csup\u003e+\u003c/sup\u003e glial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Following toxin injection, there was infiltration of small Nissl\u003csup\u003e+\u003c/sup\u003e glial cells in the corpus callosum, particularly at DPI-1 in all toxin groups. This infiltration was significantly more pronounced in the LPC and LPC\u0026thinsp;+\u0026thinsp;LPS groups at DPI-3, and only the LPC\u0026thinsp;+\u0026thinsp;LPS group showed increase in small Nissl-positive cells and loss of large Nissl\u003csup\u003e+\u003c/sup\u003e neurons at DPI-28, indicating significant persistent neuro-inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2. LPC\u0026thinsp;+\u0026thinsp;LPS induced motor incoordination, loss of myelinated fibers and neuronal loss at chronic phase of focal demyelination in spinal cord\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNeuromuscular strength was also assessed after focal injection in spinal cord using the hanging wire test. A significant decrease in latency to fall was observed at DPI-1 in the LPS group compared to the Sham group. However, this effect was not sustained a later time point in any of the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Motor coordination, assessed via the rotarod test, showed significant impairments in the LPC group at all-time points compared to Sham group. In the LPS group, significant impairment was observed only at DPI-7. In the LPC\u0026thinsp;+\u0026thinsp;LPS group, motor coordination was significantly impaired from DPI-3 to DPI-28, indicating a long-term effect on motor function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLFB and Nissl staining were used to examine structural changes in the spinal cord. The Sham group displayed normal white matter and myelin sheath (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). At DPI-1, the LPS group showed marked vacuoles at the injection site, while the LPC and LPC\u0026thinsp;+\u0026thinsp;LPS groups showed complete loss of myelinated fibres (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). However significant myelin loss was observed on DPI-3 across all the groups. At DPI-7, the LPC group was identified by the formation of distinct vacuoles, while the LPS and LPC\u0026thinsp;+\u0026thinsp;LPS groups were identified by the disarray of nerve fibres. By DPI-28, the LPS groups showed some recovery with increased myelin density, but LPS group shows disarrays of nerve fibres whereas LPC\u0026thinsp;+\u0026thinsp;LPS group showed notable vacuole formation and continued myelin loss (supplementary Fig.\u0026nbsp;2). Nissl staining indicated a distinct demarcation between white and grey matter in the Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), with fewer Nissl bodies in the ventral white and no signs of glial proliferation, chromatolysis, or neuronal apoptosis. All injection groups showed a significant increase in small Nissl\u003csup\u003e+\u003c/sup\u003e glial cells. However, the combination group (LPC\u0026thinsp;+\u0026thinsp;LPS) displayed the highest infiltration, blurring the distinction between white and grey matter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Combination of LPC\u0026thinsp;+\u0026thinsp;LPS compared to LPC or LPS is able to induce sustained demyelination in brain and spinal cord\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eWe further examined the temporal dynamics of demyelination and remyelination by focussing on MBP expression. Sham group represents the highest MBP fluorescence intensity in brain and spinal cord. In brain, LPC, LPS and LPC\u0026thinsp;+\u0026thinsp;LPS group showed significant reduction of MBP compared to sham and extent of demyelination was not significant among all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In spinal cord, LPC, LPS and especially LPC\u0026thinsp;+\u0026thinsp;LPS groups showed significant MBP reduction at DPI-1, 3, and 7. At DPI-28 LPC\u0026thinsp;+\u0026thinsp;LPS showed highest reduction even significant than LPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Pronounced activation of astrocyte and endothelial cells in combination of LPC\u0026thinsp;+\u0026thinsp;LPS toxins\u003c/h2\u003e \u003cp\u003eAstrogliosis, indicated by the presence of reactive astrocytes, were assessed in relation to blood vessel integrity, which is critical for maintaining the BBB/BSCB. To investigate this, we examined the expression of astrocytes and blood vessels in the brain and spinal cord following focal demyelination induced by LPC, LPS, and a combination of LPC and LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In brain, the expression of GFAP is dramatically high compared to sham, LPC and LPS groups signifies the activated state of astroglia. Next the expression of CD146 was highly upregulated at day-1 in LPC\u0026thinsp;+\u0026thinsp;LPS group compared to the sham, LPC and LPC\u0026thinsp;+\u0026thinsp;LPS groups followed by moderate increase. This signifies the endothelial activation preceds the astrocytic activation in combination group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn spinal cord, we have found the increased expression of GFAP over time, especially in LPC\u0026thinsp;+\u0026thinsp;LPS group significantly at DPI-28. Next, we have found upregulated expression of CD146 in LPS and LPC\u0026thinsp;+\u0026thinsp;LPS groups but not in alone LPC group. Moreover, in LPC\u0026thinsp;+\u0026thinsp;LPS the expression was significantly high compared to LPC alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). This suggests the significant activation of astrocytic and endothelial cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 LPC\u0026thinsp;+\u0026thinsp;LPS induced neuroinflammatory chemokine on reactive microglia at later phase of focal demyelination\u003c/h2\u003e \u003cp\u003eC-X-C motif chemokine receptor 1 (CX3CR1), a chemokine receptor on microglia, was assessed as a marker of neuroinflammation.\u003c/p\u003e \u003cp\u003eIn brain, Iba-1 expression is markedly increased in the LPC\u0026thinsp;+\u0026thinsp;LPS group at DPI-3, indicating the robust microglial activation. LPC and LPS showed mild increase in Iba-1 expression. CX3CR1 expression is significantly upregulated in LPC\u0026thinsp;+\u0026thinsp;LPS group at DPI-7 and normalised upto DPI-28. LPC and LPS groups alone showed moderate increase in CX3CR1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn spinal cord, the Iba-1 expression was markedly increased in LPC\u0026thinsp;+\u0026thinsp;LPS group, peaking at DPI-3. Microglia were found to be upregulated in LPC and LPS groups alone but not significantly. CX3CR1 expression is significantly upregulated in all groups but highly expressed at DPI-3 and 28. LPC and LPS groups also showed the dynamic and time dependent significant upregulated expression. This suggests that rapid microglia activation and proliferation in response to demyelination and inflammation. Sustained increased expression of CX3CR1 suggested the recruitment of microglia towards the chronic inflammatory state (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF)\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMyelination is essential for the health of neurons, as the myelin sheath facilitates rapid signal conduction along axons and maintains structural integrity. Damage to the myelin sheath leads to demyelination, often due to immune system attacks and neuroinflammation (Abbott et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Numerous studies have investigated demyelination in various neuroinflammatory diseases, such as multiple sclerosis, traumatic injuries, and neurodegeneration (Haider et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kulkarni et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zirngibl et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, replicating the complex pathology of demyelination in animal models has been challenging. Existing toxin models, such as LPC, LPS and ethidium bromide for focal and diffuse demyelination, and cuprizone and EAE for global demyelination, do not fully capture the complexity of demyelination (Lassmann and Bradl, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gharagozloo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To address this gap, we developed a model using a combination of LPC and LPS injections into the corpus callosum and the thoracic region of the spinal cord. LPC disrupts the cell membrane, increasing its permeability and allowing chemokines to infiltrate, causing localized myelin damage. LPS, through toll-like receptor activation, elicits a strong immune response.\u003c/p\u003e \u003cp\u003eIn our study, the combined effect of LPC and LPS injections in the corpus callosum and spinal cord led to neuromuscular strength impairment and motor coordination deficits. Histological analyses with LFB revealed disorganized nerve fibers, vacuole formation. Nissl staining revealed infiltration of small nissl\u003csup\u003e+\u003c/sup\u003e bodies. While remyelination typically follows demyelination in toxin models, our findings indicated a delayed remyelination phase in the LPC\u0026thinsp;+\u0026thinsp;LPS group, suggesting persistent demyelination in both the brain and spinal cord.\u003c/p\u003e \u003cp\u003eInflammatory conditions associated with demyelination can significantly impact the integrity of the blood-brain barrier (BBB) (Kirk et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zierfuss et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), blood-spinal cord barrier (BSCB) (Aub\u0026eacute; et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and modulates glial cells activity (McQuaid et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In mammals and most vertebrates, BBB/BSCB is formed by endothelial cells connected by tight junctions, providing high electrical resistance and safeguarding neurons (Abbott et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bartanusz et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Disruption of endothelial cells can damage the BBB/BSCB, enhancing the trafficking of immune cells and neuroinflammation, resulting in the degradation of the myelin sheath and demyelination (Berghoff et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Goyal and Kumar, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Studies have shown that brain endothelial cells increase the proliferation of oligodendrocyte precursor cells, which are the precursors to mature oligodendrocytes involved in myelination (Arai and Lo, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). During the stages of BBB induction and maturation, melanoma cell adhesion molecules, growth factors, and their receptors exhibit dynamic expression patterns in the cerebrovascular, coordinating interactions between endothelial cells and pericytes and orchestrating the spatiotemporal development of the BBB (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Raj et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In neuroinflammatory conditions such as multiple sclerosis (MS), CD146 gets upregulated on BBB endothelial cells, promoting the transmigration of inflammatory cells into the CNS due to BBB dysfunction (Arai and Lo, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrior studies have shown that BBB/BSCB hyperpermeability and vascular dysfunction are significant contributors to multiple sclerosis (Berghoff et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cashion et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our data demonstrated a disrupted vascular integrity and marked astrogliosis in the spinal cord and brain, particularly in the LPC\u0026thinsp;+\u0026thinsp;LPS group. These data suggests that the unique environment of the BSCB, which is more permeable than the BBB, may account for the differential impact observed between the spinal cord and brain. In case of CC demyelination endothelial dysfunction precedes the astrogliosis suggesting the causal factor for delayed remyelination. Subsequently in spinal cord demyelination, our data suggest that astrogliosis is pronounced in later phase of demyelination in LPC\u0026thinsp;+\u0026thinsp;LPS and LPC group, however the endothelial dysfunction was dynamic and significantly high than other groups. The differential expression of vascular dysfunction and astrogliosis in the brain and spinal cord can be explained by the unique environment of the BBB and BSCB. A report highlighted that BSCB is highly permeable to a variety of substances, such as TNF-α, inulin, and mannitol (Daniel et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Pan et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Another report highlighted that spinal cord pericyte coverage is less than brain pericyte coverage, thus contributing to higher permeability. Along with this, tight junction proteins such as zona occludens-1 and occludin are also less common than in the brain (Winkler et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, in our study, it is possible that the combination group of toxins has a greater impact on vascular integrity, hyperpermeability, and astrogliosis.\u003c/p\u003e \u003cp\u003eChronic activation of glial cells, such as microglia and astrocytes, is also observed in MS. These cells release various pro-inflammatory cytokines and chemokines, including TNF-α, IL-1β, CCL2, CCL5, and CXCL12. The binding of these molecules to their respective receptors aggravates the process of demyelination (Ponath et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Voet et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Healy et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A study suggested that resident microglia are crucial for the maintenance of myelin integrity (McNamara et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, another study highlighted that microglia adapt the phagocytosis-enhanced phenotype during demyelinated lesions and play a central role in neuroinflammation (Slobodov et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Consistent with this, previous studies demonstrated that the dynamic response of microglia aggravates demyelination (Chu et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, the lesion-associated microglia are more prominent than the infiltrating macrophages in the CNS following the LPC model of demyelination (Plemel et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Another study suggested that microglia markedly increased the numbers and expanded by releasing cytokine colony stimulating factor during the demyelination following the cuprizone model. However, the number decreased after pre-depleting microglia with cytokine inhibitors (Marzan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Neuron-derived chemokine fractalkine binds to the CX3CR1 receptor on microglia. However, the defective signalling of CX3CR1 and its ligand fractalkine aggravates neuroinflammation and demyelination (Mendiola et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A previous study highlighted that mRNA expression of CX3CR1 increases at later time points after SCI and mice deficient CX3CR1 gene recovered from SCI compared to the wild-type mice (Donnelly et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Our data indicated that Iba-1 expression was highly expressed at acute phase of DPI-3, more pronounced in LPC\u0026thinsp;+\u0026thinsp;LPS group. The expression of CX3CR1 was sustained and expressed throughout in case of spinal cord promoting sustained demyelination. These results suggests that LPC\u0026thinsp;+\u0026thinsp;LPS exacerbates the vascular and glial pathology, highlighting the importance of vascular-glial interactions in demyelinating diseases.\u003c/p\u003e \u003cp\u003eDespite the novelty of these findings in the LPC\u0026thinsp;+\u0026thinsp;LPS group, there are certain limitations that need to be addressed in future research. Further, we need to explore the downstream pathways to establish the synergistic effect of the combination group on demyelination. Secondly, we need to see the effect of combined toxins in oligodendrocyte development and myelination. Next, we have to provide some major evidence that the combination group has a greater impact on the spinal cord microenvironment than the brain.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study provides a comprehensive understanding of the mechanisms underlying persistent demyelination. The synergistic effects of LPC and LPS induced a cascade of events, including motor dysfunction, neuronal loss, persistent demyelination, and neuroinflammation characterized by astrogliosis, endothelial dysfunction, and microglial activation. This novel model offers a valuable platform for studying the complex interplay between myelin damage and immune activation and their contribution to persistent demyelination. By identifying these key pathological processes, our findings may facilitate the development of innovative therapeutic strategies aimed at promoting remyelination and mitigating neuroinflammation.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eDG: Investigation, Data curation, Writing and editing the draft.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKD: Investigation, Data curation and project administration.\u003c/li\u003e\n \u003cli\u003eSR: Investigation, Data curation and project administration.\u003c/li\u003e\n \u003cli\u003ePM: Investigation and project administration.\u003c/li\u003e\n \u003cli\u003eRPN: Analysis and editing of the draft.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNS: Literature, designing and editing of the manuscript.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHK: Concepts and design of study, supervision, visualization, review and editing of the final draft.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India and NIPER Ahmedabad.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current work was supported by the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India and NIPER Ahmedabad. The illustrations were prepared using the paid version of Biorender.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ (2010) Structure and function of the blood\u0026ndash;brain barrier. Neurobiology of disease 37:13-25.\u003c/li\u003e\n\u003cli\u003eArai K, Lo EH (2009) An oligovascular niche: cerebral endothelial cells promote the survival and proliferation of oligodendrocyte precursor cells. 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Molecular Neurodegeneration 17:34.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lipopolysaccharide, Lysolecithin, Spinal cord, Focal demyelination, Corpus callosum, Remyelination","lastPublishedDoi":"10.21203/rs.3.rs-8517400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8517400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFocal demyelination refers to localized loss of myelin sheath surrounds the nerve fibers. It impairs the efficient transmission of nerve impulses results in neurological symptoms. Our goal is to investigate the differential effects of lysolecithin (LPC) and lipopolysaccharides (LPS) on demyelinating processes of central nervous system. We established a novel demyelinating model using a combination of LPC and LPS with their differential effects in the corpus callosum of brain and the thoracic region of the spinal cord. To confirm the pattern of demyelination, behavioural analysis was performed. Further, brain and spinal cord samples were collected at Day-post injection-1, 3, 7, and 28. The extent of demyelination and the presence of Nissl\u003csup\u003e+\u003c/sup\u003e cells were assessed using Luxol fast blue and Nissl staining respectively. Immunofluorescence studies were done to examine demyelination and its impact on endothelial cells dysfunction, astrogliosis, and activated microglia. Our data revealed that the combination group exhibited remarkable demyelination compared to the LPC and LPS groups alone. Additionally, vascular dysfunction, astrogliosis, and activated microglia were more pronounced in the combination group. Moreover, we found much of these effects were most promising in the spinal cord compared to corpus callosum, suggesting the presence of compensatory mechanisms and a unique brain microenvironment. This study is the first to demonstrate the integrated effects of LPC and LPS toxins compared to their individual effects in both the brain and spinal cord.\u003c/p\u003e","manuscriptTitle":"Regional Vulnerability to Demyelination: A Comparative Study of the Effects of LPC, LPS, and Combined Toxins in Corpus Callosum and Spinal Cord","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-17 15:43:55","doi":"10.21203/rs.3.rs-8517400/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9adf7236-5822-487d-ba6a-f2520d595f69","owner":[],"postedDate":"February 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-21T15:25:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-17 15:43:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8517400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8517400","identity":"rs-8517400","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}