Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study

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Logan-Wesley, Karen M. Gorse, Audrey D. Lafrenaye This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4713316/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Traumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons following brain injury in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal segments, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals that sustained a TBI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation. Biological sciences/Neuroscience/Glial biology/Microglia Health sciences/Neurology/Neurological disorders/Brain injuries Microglial process convergence Traumatic brain injury Axonal injury postmortem tissue Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Traumatic brain injury (TBI) affects an estimated sixty-nine million people globally each year[ 1 ]. In 2022 alone, over twenty thousand United States service members from the Army, Navy, Air Force, and Marines, suffered from a TBI[ 2 ]. Approximately 80% of all TBI cases are classified as mild, in which diffuse pathologies that are difficult to discern via molecular imaging prevail. One of the pathological hallmarks of mild TBI is diffuse axonal injury, wherein axons are disrupted over time and progress to disconnection resulting in a proximal axonal swelling that is still connected to the neuronal soma and a distal axonal segment that degenerates via Wallerian degradation[ 3 ]. Additionally, microglia, the innate immune cell of the central nervous system, have been shown to be activated following TBI in both humans[ 4 – 9 ] and animals[ 10 – 14 ] and have been linked to cognitive changes following TBI[ 5 ]. Activated microglia fall on a spectrum from pro-inflammatory to anti-inflammatory with functions that can promote tissue neurodegeneration or neuroprotection[ 15 – 21 ]. While previous studies have identified various pro and anti-inflammatory pathways upregulated following TBI, non-phagocytic physical interactions between activated microglia and adjacent neurons have only recently begun to be investigated[ 10 , 22 – 29 ]. Previous studies from our group using a micropig model of TBI found microglial processes converging onto the injured proximal axonal segment in a phenomenon called microglial process convergence (MPC)[ 27 , 30 ]. This MPC does not appear to involve phagocytosis[ 27 ] and was not found in our rat model of TBI[ 26 ]. Specifically, in pigs, the number of activated microglial processes contacting injured proximal axonal swellings was nearly twice that observed for non-injured myelinated fibers at 6hrs following a diffuse TBI generated using the central fluid percussion injury model[ 27 ]. This MPC significantly increased from 6hrs to 1 day post injury[ 26 ]. However, in rats, there were far fewer microglial processes contacting injured axonal swellings compared to non-injured myelinated fibers following TBI, indicating that MPC might be a phenomenon associated with higher order gyrencephalic brains[ 26 ]. To investigate the potential that TBI-induced MPC onto injured axons occurs in the human brain, in the current study we quantitatively assessed the prevalence of MPC onto injured axonal swellings and intact axonal segments in human postmortem brain tissue. Results Microglial Process Convergence Increases Following Traumatic Brain Injury and Axonal Injury To investigate the potential for microglial process convergence occurring on injured axonal swellings or intact myelinated fibers in the human brain, multiplexed immunohistochemistry against APP to visualize injured axons, MBP to visualize intact myelinated axons, and Iba-1 to visualize microglia processes was done on human postmortem tissue from the DoD/USU tissue repository. When all fibers that were analyzed across all cases were collated as APP + injured axonal swelling or MBP + intact myelinated fibers, it was found that more Iba-1 + microglial processes/um of the perimeter were in direct contact with APP + axonal swellings compared to MBP + intact myelinated fibers (Fig. 1 ; U = 32,240, p = 2.4X10 − 4 ). The paraffin sections were ~ 5µm thick, precluding the ability to perform 3D reconstructions of the axonal swellings, as we had done for our previous studies[ 26 , 27 ]. As a single 2D image of the axonal segments is likely to be missing processes that are out of the plan of section, we also investigated the number of Iba-1 + microglial processes that were within 5um of the axonal segments. More microglial processes were found within 5um of APP + axonal swellings compared to MBP + intact myelinated fibers (Fig. 1 ; U = 34,260, p = 2.99X10 − 6 ), indicating that more microglial processes are close to the injured axonal swellings. After completing this initial analysis of the overall comparison between APP + swellings and MBP + myelinated fibers, the cases were un-blinded. Following case unblinding, it was discovered that some APP + axonal swellings were identified in control individuals and some MBP + myelinated fibers were analyzed from individuals that suffered a TBI (Fig. 2 ). Therefore, the analyzed fibers were organized into four groups: 1) MBP + intact myelinated fibers in control tissue, 2) MBP + intact myelinated fibers in TBI tissue, 3) APP + axonal swellings in control tissue, and 4) APP + axonal swellings in TBI tissue. When the data was stratified by axonal injury and TBI the finding of more microglial process convergence occurring directly onto APP + injured axonal swellings, specifically within TBI tissue (χ 2 (3) = 15.53, p = 0.001; Fig. 3 ). There were also significantly more microglia processes within 5µm of APP + injured swellings in both control and TBI tissue as compared to MBP + fibers in either injured or control postmortem samples was maintained (χ 2 (3) = 21.97, p = 6.61X10 − 5 ; Fig. 3 ). The morphology between MBP + intact axonal segments and APP + axonal swellings was significantly different (χ 2 (3) = 12.2, p = 0.007), with the APP + axonal swellings within TBI tissue having smaller perimeters compared to the MBP + intact axonal fibers within control tissue (p = 0.003). The APP + axonal swellings in both control and TBI tissue had significantly higher circularity indices compared to the MBP + axonal fibers in either control or TBI tissue (χ 2 (3) = 332.35, p = < 0.001; Fig. 4 ). While the MBP + intact axonal fibers within TBI tissue were more circular than the MBP + axonal fibers in control tissue (p < 0.001), the morphology of APP + axonal swellings within control cases, however, were consistent with the APP + axonal swellings in TBI cases (Fig. 4 ; Perimeter p = 0.08; circularity p = 0.83). To validate the by eye counts of Iba-1 microglial process puncta onto the APP + axonal swellings, the integrated density/intensity of Iba-1 microglial processes in the region of the APP + axonal swelling or MBP + myelinated fiber was assessed. The intensity of Iba-1 labeling was significantly higher in APP + injured axonal swellings compared to control MBP + axons (χ 2 (3) = 56.217, p = 3.78X10 − 12 ; Fig. 5 ). Specifically, there were not differences in the intensity of Iba-1 between APP + injured axons (p = 0.72) nor between MBP + axonal segments (p = 0.99) within control tissue compared to TBI tissue. There was significantly higher Iba-1 integrated density within APP + axonal swellings compared to MBP + intact axonal segments regardless of TBI status of the case (MBP + intact fiber in control cases vs. APP + swellings in control cases p < 0.001; MBP + intact fiber in TBI cases vs. APP + swellings in TBI cases p < 0.001; Fig. 5 ). This finding validated the counts of microglial process convergence onto injured axonal swelling, demonstrating increases in microglial process convergence onto APP + injured axonal swellings in the human brain. Discussion The current study demonstrates that microglial processes converge onto injured axonal swellings in the human brain following TBI. The perimeters of the axonal segments were relatively consistent across non-injured MBP + myelinated fibers in both control and TBI tissue as well as APP + injured axonal swellings within control tissue. The APP + injured axonal swellings within TBI tissue had significantly lower perimeters than the MBP + intact myelinated axonal segments within control tissue. The number of Iba-1 + microglial processes in direct contact with the axonal segment, however, was significantly higher onto APP + axonal swellings compared to MBP + non-injured axonal segments, despite the lower perimeter available for contact. There were also significantly more microglial processes within 5µm of the injured axons compared to the non-injured MBP + axonal segments. Our group previously observed MPC in the micropig brain acutely following a central fluid percussion diffuse TBI[ 27 ]. This phenomenon; however, was not recapitulated following TBI in the rat[ 26 ], indicating that MPC onto injured axons might be species specific. The current findings that MPC onto injured axonal swellings occurs in human postmortem tissue, validates our previous findings in the micro pig and indicates that MPC following TBI might primarily manifest in higher order gyrencephalic brains. These findings show that MPC is a phenomenon that occurs in the human population, necessitating further investigation. Diffuse axonal injury (DAI) or traumatic axonal injury (TAI) is one of the hallmark pathologies of mild TBI[ 31 – 36 ]. It was originally thought that DAI occurs following TBI due to the mechanical shearing of the axons. However, this is only true for a subset of axons that are observed to be injured within minutes of TBI, which is referred to as primary axotomy. Rather, secondary axotomy/axonal injury, which occurs sub-acutely after injury in rodent models of TBI is the phenomenon that is typically investigated. Secondary axonal injury involves accumulation of the cytoskeleton and organelles within the axons[ 37 – 39 ]. Specifically, the tensile forces of the TBI cause axonal alterations that allow an influx of calcium into the axon. This calcium influx leads to activation of cysteine protease pathways, which leads to degradation of the cytoskeleton and ultimately lead to a reactive axonal swelling as anterogradely transported proteins and organelles pool at the end of the proximal axon[ 38 – 40 ]. In the early 1990s immunohistochemistry against the anterogradely transported protein, APP, was found to efficiently label the proximal injured axonal segment where it pooled[ 41 , 42 ]. Immunohistochemical labeling of APP has since become the gold standard for identifying DAI pathologically. Secondary axonal injury is typically studied in rodents 6–24 hours following injury, when it is most prevalent[ 43 , 44 ]. Within higher order animals DAI appears to be prevalent starting hours following injury and peaking at 1w post-TBI[ 13 ], however DAI has been shown to last up to 6 months in a pig model of TBI[ 45 ] and has been observed in human postmortem tissue from people several years following a TBI[ 46 , 47 ]. Many recent studies have demonstrated the impact of inflammatory cascades in regulating behavioral morbidities, general pathology, and neuronal function in both the normal brain and in various disease states, including TBI[ 21 , 48 , 49 ]. Neuroinflammation has been demonstrated in various brain regions in the human population chronically following TBI[ 5 , 9 , 20 , 50 ]. Microglia, the innate immune cells of the brain, are critical mediators of these TBI-induced neuroinflammatory processes[ 16 , 51 – 57 ]. Microglia have been shown to contact specific areas of the axon in the mouse brain during homeostasis including, the nodes of Ranvier[ 58 ], the axon initial segment[ 10 ], synapses[ 59 ], and neuronal soma[ 60 ]. Many studies using rodents have indicated that reduction or elimination of activated microglia and/or targeting various neuroinflammatory signaling pathways ameliorates downstream pathology and behavioral morbidity[ 24 , 61 – 64 ]. Conversely, other studies have also found that anti-inflammatory microglial activation is necessary and potentially advantageous[ 17 , 54 , 65 – 70 ]. These studies demonstrated activated microglia can secrete neurotrophic factors[ 15 , 71 – 73 ], which would suggest a potential ameliorative effect of microglia following injury in some cases. Additionally, recent studies have shown that microglia physical contacts play a role in regulating neuronal activity, either increasing activity following anesthesia[ 74 , 75 ] or decreasing activity following epileptiform activity[ 60 , 67 ]. A study by Schirmer et. al. investigating human post-mortem tissue found a significant positive correlation between the density of microglia and axonal outgrowth as well as the duration of patient survival following TBI, however, they did not quantitatively investigate the physical interactions between microglia and injured axons[ 76 ]. Another study done in vitro and in rats following a spinal cord injury found that exosomes from anti-inflammatory microglia increased neurite outgrowth in vitro and increased GAP43 expression in vivo, indicating that microglia could play a role in axonal outgrowth[ 17 ]. Microglia have been observed within physical proximity to injured axonal swellings in human postmortem tissue[ 76 – 78 ]. Oehmichen et al. observed an increase in CD-68 + microglial cells areas of axonal injury in the white matter at least five days post-TBI in human postmortem tissue, however, they only observed limited physical interactions between the CD68 + cells and the APP + axonal segments[ 77 ]. Ryu et al. qualitatively identified areas in which Iba-1 + microglia were in proximity to APP + injured axons in postmortem tissue from individuals following both motor vehicle accident and blast induced TBI[ 78 ]. Although, these previous studies indicated that there could be direct physical interactions between microglia and injured axonal segments, our current study is the first to quantitatively show that MPC occurs onto injured proximal axonal segments in human brain tissue following TBI. We do appreciate that there are limitations to the current study, mainly, that all cases were from male doners. There is evidence that males and females respond to TBI differently[ 79 ]. A recent study also found that the burden of axonal injury following a diffuse TBI in a pig model was significantly higher in females compared to age matched males[ 80 ]. Further, microglia have been shown to be different in males and females[ 81 – 85 ]. Therefore, investigations into MPC in both the male and female population should be done to fully appreciate the prevalence of MPC onto injury axons in the human brain. Additionally, these sections were only 15µm thick, precluding a 3D investigation of MPC, as was done in our previous micro pig studies[ 26 , 27 ]. It is likely that the numbers of microglial processes we found converging onto the axonal segments in the current study were artificially lower than they might actually be due to the section thickness. Therefore, investigations using thicker tissue in which 3D reconstructions could be done, would be warranted to glean a better appreciation of the degree of MPC onto injured axonal swellings in human postmortem tissue of both males and females. Despite these limitations, the current study is the first to quantitatively demonstrate MPC onto injured axons in the human brain following TBI. These findings indicate that MPC is a component of human TBI and that further studies exploring the phenotypes and overall roles of the microglia involved in MPC following TBI should be investigated. Methods Samples Human brain samples were acquired from the Department of Defense (DoD)/Uniform Services University (USU) Tissue Repository. All cases were from males between 26–69 years old (median age of 36 years) with a maximum postmortem interval of 1day. All identifiers were removed from the samples. Tissue was paraffin embedded and sectioned at 15um. Slides containing areas demonstrating axonal injury regardless of brain region were used for this study. A total of 11 human brain samples were used. Of these samples, 6 were confirmed TBI cases with demonstrated areas of axonal injury and 5 were controls. All study staff was blinded to case group throughout the labeling, imaging, and analysis. Immunohistochemistry To visualize the interactions between microglia and injured or intact axonal segments in the human brain multiplexed fluorescent immunohistochemistry was performed. To identify injured axons, an antibody against amyloid precursor protein (APP) was used, which indicates axonal transport issues indicative of axonal injury [ 13 , 14 , 58 ]. To visualize microglia, an antibody against ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Intact, non-injured axonal fibers were visualized using an antibody against myelin basic protein (MBP). In this procedure, sections were deparaffinized by incubating slides in progressively more concentrated alcohols. Antigen retrieval was done by steaming the tissue in pH 6.0 citric acid buffer for 30min. Tissue was then blocked and permeabilized at room temperature in 5% normal goat serum (NGS), 2% bovine serum albumin (BSA) and 1.5% triton in phosphate buffered saline for 2hr followed by overnight incubation with a rabbit antibody against microglial Iba-1 (1:200; Cat.#019-19741 Wako; Richmond, VA, USA) at 4C° in 5% NGS/2% BSA/0.5% triton. Tissue was washed with 1%NGS/1%BSA in PBS at least six times prior to secondary antibody incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:700; Cat.# A- 11008, Life Technologies, Carlsbad, CA, USA) in 1%NGS/1%BSA/PBS at room temperature for 2hr. Tissue was washed in PBS at least four times prior to overnight incubation with a mouse antibody against the 22C11 clone of APP (1:200; Cat.#14-9749-82, ThermoFisher Scientific, Waltham, MA, USA) in 5% NGS/2% BSA/0.5% triton at 4C°. Tissue was washed with 1%NGS/1%BSA in PBS at least six times prior to the next secondary antibody incubation with Alexa Fluor 647-conjugated goat anti-mouse IgG (1:700; Cat.# A- 21237, Life Technologies, Carlsbad, CA, USA) in 1%NGS/1%BSA/PBS at room temperature for 2hr. Iba-1 and APP labeled tissue was washed in PBS at least four times prior to overnight incubation with a rat antibody against MBP (1:200; Novus) at 4C° in 5% NGS/2% BSA/0.5% triton. Tissue was washed with 1%NGS/1%BSA in PBS at least six times prior to the third secondary antibody incubation with Alexa Fluor 568-conjugated goat anti-rat IgG (1:700; Cat.# A- 11077, Life Technologies, Carlsbad, CA, USA) in 1%NGS/1%BSA/PBS at room temperature for 2hr. Tissue was washed in PBS at least four times. Multiplex labeled tissue was coversliped with Vectashield hard-set mounting medium with Dapi (Cat.#H-1500; Vector Laboratories, Burlingame, CA, USA). Microglial Process Convergence Analysis The fluorescently immunolabeled slides were imaged on the Keyence BZ-X800 microscope (Keyence Corporation of America, Itasca, IL, USA) at 40X magnification. One section was analyzed for each case by an investigator blinded to group. A navigation super-image was generated using the far-red channel in which the APP + injured axonal swellings could be visualized. Images containing at least 1 APP + injured axonal swellings were captured or no APP + swellings but clean MBP labeling were captured. Fewer MBP only images were captured, as there were several analyzable MBP + intact myelinated fibers in each captured image, whereas there were few analyzable APP + axonal swellings in each captured image. At least 25 images were taken for most cases, however, only 13 images were captured for 1 case as no APP + swellings were identified. Across all samples a total of 161 non-injured axonal segments from controls, 36 non-injured axonal segments from injured samples, 105 injured axonal swellings from controls, and 173 injured axonal swellings from injured samples were analyzed for the current study. Fiji Image J software (National Institute of Health, Bethesda, MD, USA) was used to evaluate the 2D images. Image scales were set to 5.3 pixels/um To assess the interaction between microglia and injured axonal swellings, the APP + axonal swelling was traced using the freehand tool and measured for perimeter, area, shape descriptors (aspect ratio, circularity, round, solidity), integrated density, and mean grey value. The number of microglial processes and puncta that were directly touching the APP + axonal swelling was counted by hand. Then, the region encircling the APP + axonal swelling was enlarged by 5um and the microglial processes and puncta within the enlarged region was counted by hand. In order to visualize the interaction between microglia and intact axonal segments, a random number generator was used to generate x,y coordinates to choose a MBP + axonal segment on the image. The axonal segment was traced with the freehand tool and measured for perimeter, area, shape descriptors (aspect ratio, circularity, roundness, and solidity), integrated density, and mean grey value. The number of microglial processes and puncta that were directly touching the MBP + intact axonal segment was counted by hand. Then, the region encircling the axonal segment was enlarged by 5um and the number of microglial processes and puncta within the enlarged region were counted by hand. Statistical Analysis The statistics were run using IBM SPSS software (IBM Corp., Armonk, NY). A Shapiro-Wilk test was conducted to test for normality of the data. As the data was not normally distributed, a Mann-Whitney U test was used to test differences between all APP + injured axonal swellings and all MBP + intact axonal segments. A Kruskal-Wallis test was run to assess differences across multiple groups. A Bonferroni post hoc was used to correct for multiple pairwise comparisons. Statistical significance was set to a p -value of < 0.05. Data is presented as means and standard error of the mean. All raw data is included in Supplemental table 1 . Declarations Acknowledgments This work would not have been possible without the tissue from the Department of Defense (DoD)/Uniform Services University (USU) Tissue Repository and Neuropathology Core, which is funded by the Department of Defense and is housed within the Center for Neuroscience and Regenerative Medicine. Authors’ Contributions ALW performed quantitative analysis and wrote the first draft of the manuscript. KG captured images for analysis and performed quantitative analysis. AL captured images for analysis, organized the data, edited the manuscript, managed the project, and secured funding for the completion of this study. Competing interests The authors declare that they have no competing interests. Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Following publication these data will also be available on the Open Science Framework along with the analysis protocol. Approval for human experiments The current study was reviewed by the Virginia Commonwealth University Institutional Review Board under IRB ID HM20029279 and was determined not to be research involving human subjects as defined by DHHS and FDA regulations. Funding This work was funded by NINDS grants R21 NS126611 and R01 NS128104. References Dewan MC, Rattani A, Gupta S, Baticulon RE, Hung Y-C, Punchak M, et al. 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Activation of Neuronal NMDA Receptors Triggers Transient ATP-Mediated Microglial Process Outgrowth. Journal of Neuroscience. 2014;34:10511–27. Eyo UB, Peng J, Swiatkowski P, Mukherjee A, Bispo A, Wu L-J. Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2014;34:10528–40. Franco ECS, Cardoso MM, Gouvêia A, Pereira A, Gomes-Leal W. Modulation of microglial activation enhances neuroprotection and functional recovery derived from bone marrow mononuclear cell transplantation after cortical ischemia. Neuroscience Research. 2012;73:122–32. Hanlon LA, Raghupathi R, Huh JW. Depletion of microglia immediately following traumatic brain injury in the pediatric rat: Implications for cellular and behavioral pathology. Experimental Neurology. 2019;316:39–51. Wang C, Ji Y, Zhang H, Ye Y, Zhang G, Zhang S, et al. Increased level of exosomal miR-20b-5p derived from hypothermia-treated microglia promotes neurite outgrowth and synapse recovery after traumatic brain injury. Neurobiology of Disease. 2023;179:106042. Batchelor PE, Porritt MJ, Martinello P, Parish CL, Liberatore GT, Donnan G a, et al. Macrophages and Microglia Produce Local Trophic Gradients That Stimulate Axonal Sprouting Toward but Not beyond the Wound Edge. Molecular and cellular neurosciences. 2002;21:436–53. Parkhurst CN, Yang G, Ninan I, Savas JN, Yates JR, Lafaille JJ, et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell. 2013;155:1596–609. Dougherty KD, Dreyfus CF, Black IB. Brain-derived neurotrophic factor in astrocytes, oligodendrocytes, and microglia/macrophages after spinal cord injury. Neurobiology of disease. 2000;7:574–85. Liu YU, Ying Y, Li Y, Eyo UB, Chen T, Zheng J, et al. Neuronal network activity controls microglial process surveillance in awake mice via norepinephrine signaling. Nat Neurosci. 2019;22:1771–81. Cao K, Qiu L, Lu X, Wu W, Hu Y, Cui Z, et al. Microglia modulate general anesthesia through P2Y12 receptor. Current Biology. 2023;33:2187–2200.e6. Schirmer L, Merkler D, König FB, Brück W, Stadelmann C. Neuroaxonal regeneration is more pronounced in early multiple sclerosis than in traumatic brain injury lesions. Brain Pathology. 2013;23:2–12. Oehmichen M, Theuerkauf I, Meissner C. Is traumatic axonal injury (AI) associated with an early microglial activation? Application of a double-labeling technique for simultaneous detection of microglia and AI. Acta neuropathologica. 1999;97:491–4. Ryu J, Horkayne-Szakaly I, Xu L, Pletnikova O, Leri F, Eberhart C, et al. The problem of axonal injury in the brains of veterans with histories of blast exposure. Acta Neuropathologica Communications. 2014;2:1–14. Biegon A. Considering Biological Sex in Traumatic Brain Injury. Frontiers in Neurology. 2021;12:576366. Song H, Tomasevich A, Paolini A, Browne KD, Wofford KL, Kelley B, et al. Sex differences in the extent of acute axonal pathologies after experimental concussion. Acta Neuropathol. 2024;147:79. Yanguas-Casás N. Physiological sex differences in microglia and their relevance in neurological disorders. Neuroimmunology and Neuroinflammation. 2020;7:13–22. Martinez-Muniz GA, Wood SK. Special Section on Sexual Dimorphism in Neuroimmune Cells Sex Differences in the Inflammatory Consequences of Stress: Implications for Pharmacotherapy. THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther. 2020;375:161–74. VanRyzin JW, Pickett LA, McCarthy MM. Microglia: Driving critical periods and sexual differentiation of the brain. Developmental Neurobiology. 2018;78:580–92. Schwarz JM, Sholar PW, Bilbo SD. Sex differences in microglial colonization of the developing rat brain. Journal of Neurochemistry. 2012;120:948–63. Doran SJ, Ritzel RM, Glaser EP, Henry RJ, Faden AI, Loane DJ. Sex differences in acute neuroinflammation after experimental traumatic brain injury are mediated by infiltrating myeloid cells. Journal of Neurotrauma. 2019;36:1040–53. Additional Declarations No competing interests reported. Supplementary Files Supplementaltable1.xlsx Cite Share Download PDF Status: Published Journal Publication published 12 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviews received at journal 25 Jul, 2024 Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 15 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers invited by journal 12 Jul, 2024 Editor assigned by journal 12 Jul, 2024 Editor invited by journal 11 Jul, 2024 Submission checks completed at journal 10 Jul, 2024 First submitted to journal 09 Jul, 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-4713316","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":333016734,"identity":"388ad890-5c45-40dc-a101-59c27cde61c4","order_by":0,"name":"Amanda L. Logan-Wesley","email":"","orcid":"","institution":"Virginia Commonwealth University","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"L.","lastName":"Logan-Wesley","suffix":""},{"id":333016736,"identity":"afa3063d-a587-481f-b331-8c4378b7e328","order_by":1,"name":"Karen M. Gorse","email":"","orcid":"","institution":"Virginia Commonwealth University","correspondingAuthor":false,"prefix":"","firstName":"Karen","middleName":"M.","lastName":"Gorse","suffix":""},{"id":333016739,"identity":"b84e1909-adca-41bf-adc5-e84fe85b039a","order_by":2,"name":"Audrey D. Lafrenaye","email":"data:image/png;base64,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","orcid":"","institution":"Virginia Commonwealth University","correspondingAuthor":true,"prefix":"","firstName":"Audrey","middleName":"D.","lastName":"Lafrenaye","suffix":""}],"badges":[],"createdAt":"2024-07-09 15:54:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4713316/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4713316/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-71312-7","type":"published","date":"2024-09-12T15:58:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62157423,"identity":"c96fd3dc-8b59-40e7-a0c6-e4869a516662","added_by":"auto","created_at":"2024-08-09 21:18:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":355382,"visible":true,"origin":"","legend":"\u003cp\u003eMicroglial processes converged onto injured axons in human postmortem tissue. Box and whisker plots of Iba-1+ microglial processes A) in direct contact with or B) within 5mm of MBP+ non-injured intact axonal fibers (n=197) and APP+ injured axonal swellings (n=278). * p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/e480dc54f3247f9ad25b0198.png"},{"id":62157421,"identity":"783f1316-867e-4b46-8ca6-6291fb203b5b","added_by":"auto","created_at":"2024-08-09 21:18:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3150863,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative fluorescent micrographs of microglial process interactions with either non-injured intact myelinated axonal fibers (first two columns) or injured axonal swellings (second two columns). Nuclei were labeled with Dapi in blue (top panel). Microglia were immunolabeled with Iba-1 which is pseudo colored green in the second panel. Intact myelinated fibers immunolabeled with MBP (first two columns of the third panel) or injured axonal swellings immunolabeled with APP (last two columns of the third panel) were pseudo colored in red. The last panel shows the full overlay for the multiplex immunohistochemical labeling. Scale bar is 20mm.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/11919550d9537237d40c38ce.png"},{"id":62157425,"identity":"dbc4d362-3ae0-4046-bdec-96a443e7bf09","added_by":"auto","created_at":"2024-08-09 21:18:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":352326,"visible":true,"origin":"","legend":"\u003cp\u003eMicroglial processes appear to converge onto injured axons in control and TBI cases. Box and whisker plots of Iba-1+ microglial processes A) in direct contact with or B) within 5mm of MBP+ non-injured myelinated axonal fibers in either control cases (n=161 fibers) or TBI cases (n=36 fibers) and APP+ injured axonal swellings in control cases (n=105 swellings) or TBI cases (n=173 swellings). Note that while the APP+ axonal swellings within the control cases did not have significantly higher Iba-1+ microglial processes directly touching it (p=0.053), both control cases and TBI cases had significantly more Iba-1+ microglial processes within 5mm compared to the non-injured MBP+ fiber counterparts. * p\u0026lt;0.05 compared to MBP+ intact fibers in control tissue. # p\u0026lt;0.05 compared to MBP+ intact fibers in control tissue.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/eb3eb8e8c16b714e3e28dba6.png"},{"id":62157961,"identity":"bdd53344-81ca-43d8-8a57-be5e41d9aaab","added_by":"auto","created_at":"2024-08-09 21:26:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":397724,"visible":true,"origin":"","legend":"\u003cp\u003eInjured axons have lower perimeters and are more circular than MBP+ intact myelinated fibers. Box and whisker plots of A) the perimeter of axonal segments and B) the circularity of axonal segments of MBP+ intact axonal fibers in either control cases (n=161 fibers) or TBI cases (n=36 fibers) and APP+ injured axonal swellings in control cases (n=105 swellings) or TBI cases (n=173 swellings). Note that while there was no significant difference between APP+ axonal swellings in control tissue compared to MBP+ myelinated fibers in control tissue (p=0.08), injured APP+ axonal swellings within TBI tissue had significantly lower perimeters. * p\u0026lt;0.05 compared to MBP+ intact fibers in control tissue. # p\u0026lt;0.05 compared to MBP+ intact fibers in control tissue.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/21fb45290d823e1efc951b59.png"},{"id":62157427,"identity":"230bd4c1-ce54-4452-91e2-b1a3e1a1dafd","added_by":"auto","created_at":"2024-08-09 21:18:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":173944,"visible":true,"origin":"","legend":"\u003cp\u003eInjured axons have higher Iba-1+ microglia fluorescent intensity than MBP+ intact myelinated fibers. Box and whisker plots depicting the integrated density of Iba-1+ microglia within axonal segments of MBP+ intact axonal fibers in either control cases (n=161 fibers) or TBI cases (n=36 fibers) and APP+ injured axonal swellings in control cases (n=105 swellings) or TBI cases (n=173 swellings). Injured APP+ axonal swellings in either control or TBI tissue had significantly higher Iba-1+ fluorescent intensity within the swellings compared to intact MBP+ axonal fibers. * p\u0026lt;0.05 compared to MBP+ intact fibers in control tissue. # p\u0026lt;0.05 compared to MBP+ intact fibers in control tissue.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/d852e000a4ec37b2d02f1aa8.png"},{"id":64619412,"identity":"9031f56a-ad87-4a81-a818-b516d0774b38","added_by":"auto","created_at":"2024-09-16 16:14:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5232237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/0c30963d-9b89-4979-b0c7-34042d5e529c.pdf"},{"id":62157962,"identity":"28bbca60-7117-4c52-b094-6672e3462f56","added_by":"auto","created_at":"2024-08-09 21:26:09","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":78784,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaltable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4713316/v1/2e369f8b126c8012025cea49.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraumatic brain injury (TBI) affects an estimated sixty-nine million people globally each year[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 2022 alone, over twenty thousand United States service members from the Army, Navy, Air Force, and Marines, suffered from a TBI[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Approximately 80% of all TBI cases are classified as mild, in which diffuse pathologies that are difficult to discern via molecular imaging prevail. One of the pathological hallmarks of mild TBI is diffuse axonal injury, wherein axons are disrupted over time and progress to disconnection resulting in a proximal axonal swelling that is still connected to the neuronal soma and a distal axonal segment that degenerates via Wallerian degradation[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, microglia, the innate immune cell of the central nervous system, have been shown to be activated following TBI in both humans[\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and animals[\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and have been linked to cognitive changes following TBI[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Activated microglia fall on a spectrum from pro-inflammatory to anti-inflammatory with functions that can promote tissue neurodegeneration or neuroprotection[\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile previous studies have identified various pro and anti-inflammatory pathways upregulated following TBI, non-phagocytic physical interactions between activated microglia and adjacent neurons have only recently begun to be investigated[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous studies from our group using a micropig model of TBI found microglial processes converging onto the injured proximal axonal segment in a phenomenon called microglial process convergence (MPC)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This MPC does not appear to involve phagocytosis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and was not found in our rat model of TBI[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Specifically, in pigs, the number of activated microglial processes contacting injured proximal axonal swellings was nearly twice that observed for non-injured myelinated fibers at 6hrs following a diffuse TBI generated using the central fluid percussion injury model[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This MPC significantly increased from 6hrs to 1 day post injury[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, in rats, there were far fewer microglial processes contacting injured axonal swellings compared to non-injured myelinated fibers following TBI, indicating that MPC might be a phenomenon associated with higher order gyrencephalic brains[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To investigate the potential that TBI-induced MPC onto injured axons occurs in the human brain, in the current study we quantitatively assessed the prevalence of MPC onto injured axonal swellings and intact axonal segments in human postmortem brain tissue.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMicroglial Process Convergence Increases Following Traumatic Brain Injury and Axonal Injury\u003c/h2\u003e \u003cp\u003eTo investigate the potential for microglial process convergence occurring on injured axonal swellings or intact myelinated fibers in the human brain, multiplexed immunohistochemistry against APP to visualize injured axons, MBP to visualize intact myelinated axons, and Iba-1 to visualize microglia processes was done on human postmortem tissue from the DoD/USU tissue repository. When all fibers that were analyzed across all cases were collated as APP\u0026thinsp;+\u0026thinsp;injured axonal swelling or MBP\u0026thinsp;+\u0026thinsp;intact myelinated fibers, it was found that more Iba-1\u0026thinsp;+\u0026thinsp;microglial processes/um of the perimeter were in direct contact with APP\u0026thinsp;+\u0026thinsp;axonal swellings compared to MBP\u0026thinsp;+\u0026thinsp;intact myelinated fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; U\u0026thinsp;=\u0026thinsp;32,240, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4X10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e). The paraffin sections were ~\u0026thinsp;5\u0026micro;m thick, precluding the ability to perform 3D reconstructions of the axonal swellings, as we had done for our previous studies[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. As a single 2D image of the axonal segments is likely to be missing processes that are out of the plan of section, we also investigated the number of Iba-1\u0026thinsp;+\u0026thinsp;microglial processes that were within 5um of the axonal segments. More microglial processes were found within 5um of APP\u0026thinsp;+\u0026thinsp;axonal swellings compared to MBP\u0026thinsp;+\u0026thinsp;intact myelinated fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; U\u0026thinsp;=\u0026thinsp;34,260, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.99X10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e), indicating that more microglial processes are close to the injured axonal swellings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter completing this initial analysis of the overall comparison between APP\u0026thinsp;+\u0026thinsp;swellings and MBP\u0026thinsp;+\u0026thinsp;myelinated fibers, the cases were un-blinded. Following case unblinding, it was discovered that some APP\u0026thinsp;+\u0026thinsp;axonal swellings were identified in control individuals and some MBP\u0026thinsp;+\u0026thinsp;myelinated fibers were analyzed from individuals that suffered a TBI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, the analyzed fibers were organized into four groups: 1) MBP\u0026thinsp;+\u0026thinsp;intact myelinated fibers in control tissue, 2) MBP\u0026thinsp;+\u0026thinsp;intact myelinated fibers in TBI tissue, 3) APP\u0026thinsp;+\u0026thinsp;axonal swellings in control tissue, and 4) APP\u0026thinsp;+\u0026thinsp;axonal swellings in TBI tissue. When the data was stratified by axonal injury and TBI the finding of more microglial process convergence occurring directly onto APP\u0026thinsp;+\u0026thinsp;injured axonal swellings, specifically within TBI tissue (χ\u003csup\u003e2\u003c/sup\u003e(3)\u0026thinsp;=\u0026thinsp;15.53, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). There were also significantly more microglia processes within 5\u0026micro;m of APP\u0026thinsp;+\u0026thinsp;injured swellings in both control and TBI tissue as compared to MBP\u0026thinsp;+\u0026thinsp;fibers in either injured or control postmortem samples was maintained (χ\u003csup\u003e2\u003c/sup\u003e(3)\u0026thinsp;=\u0026thinsp;21.97, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.61X10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe morphology between MBP\u0026thinsp;+\u0026thinsp;intact axonal segments and APP\u0026thinsp;+\u0026thinsp;axonal swellings was significantly different (χ\u003csup\u003e2\u003c/sup\u003e(3)\u0026thinsp;=\u0026thinsp;12.2, p\u0026thinsp;=\u0026thinsp;0.007), with the APP\u0026thinsp;+\u0026thinsp;axonal swellings within TBI tissue having smaller perimeters compared to the MBP\u0026thinsp;+\u0026thinsp;intact axonal fibers within control tissue (p\u0026thinsp;=\u0026thinsp;0.003). The APP\u0026thinsp;+\u0026thinsp;axonal swellings in both control and TBI tissue had significantly higher circularity indices compared to the MBP\u0026thinsp;+\u0026thinsp;axonal fibers in either control or TBI tissue (χ\u003csup\u003e2\u003c/sup\u003e(3)\u0026thinsp;=\u0026thinsp;332.35, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). While the MBP\u0026thinsp;+\u0026thinsp;intact axonal fibers within TBI tissue were more circular than the MBP\u0026thinsp;+\u0026thinsp;axonal fibers in control tissue (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), the morphology of APP\u0026thinsp;+\u0026thinsp;axonal swellings within control cases, however, were consistent with the APP\u0026thinsp;+\u0026thinsp;axonal swellings in TBI cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Perimeter p\u0026thinsp;=\u0026thinsp;0.08; circularity p\u0026thinsp;=\u0026thinsp;0.83).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate the by eye counts of Iba-1 microglial process puncta onto the APP\u0026thinsp;+\u0026thinsp;axonal swellings, the integrated density/intensity of Iba-1 microglial processes in the region of the APP\u0026thinsp;+\u0026thinsp;axonal swelling or MBP\u0026thinsp;+\u0026thinsp;myelinated fiber was assessed. The intensity of Iba-1 labeling was significantly higher in APP\u0026thinsp;+\u0026thinsp;injured axonal swellings compared to control MBP\u0026thinsp;+\u0026thinsp;axons (χ\u003csup\u003e2\u003c/sup\u003e(3)\u0026thinsp;=\u0026thinsp;56.217, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.78X10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, there were not differences in the intensity of Iba-1 between APP\u0026thinsp;+\u0026thinsp;injured axons (p\u0026thinsp;=\u0026thinsp;0.72) nor between MBP\u0026thinsp;+\u0026thinsp;axonal segments (p\u0026thinsp;=\u0026thinsp;0.99) within control tissue compared to TBI tissue. There was significantly higher Iba-1 integrated density within APP\u0026thinsp;+\u0026thinsp;axonal swellings compared to MBP\u0026thinsp;+\u0026thinsp;intact axonal segments regardless of TBI status of the case (MBP\u0026thinsp;+\u0026thinsp;intact fiber in control cases vs. APP\u0026thinsp;+\u0026thinsp;swellings in control cases p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; MBP\u0026thinsp;+\u0026thinsp;intact fiber in TBI cases vs. APP\u0026thinsp;+\u0026thinsp;swellings in TBI cases p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This finding validated the counts of microglial process convergence onto injured axonal swelling, demonstrating increases in microglial process convergence onto APP\u0026thinsp;+\u0026thinsp;injured axonal swellings in the human brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current study demonstrates that microglial processes converge onto injured axonal swellings in the human brain following TBI. The perimeters of the axonal segments were relatively consistent across non-injured MBP\u0026thinsp;+\u0026thinsp;myelinated fibers in both control and TBI tissue as well as APP\u0026thinsp;+\u0026thinsp;injured axonal swellings within control tissue. The APP\u0026thinsp;+\u0026thinsp;injured axonal swellings within TBI tissue had significantly lower perimeters than the MBP\u0026thinsp;+\u0026thinsp;intact myelinated axonal segments within control tissue. The number of Iba-1\u0026thinsp;+\u0026thinsp;microglial processes in direct contact with the axonal segment, however, was significantly higher onto APP\u0026thinsp;+\u0026thinsp;axonal swellings compared to MBP\u0026thinsp;+\u0026thinsp;non-injured axonal segments, despite the lower perimeter available for contact. There were also significantly more microglial processes within 5\u0026micro;m of the injured axons compared to the non-injured MBP\u0026thinsp;+\u0026thinsp;axonal segments. Our group previously observed MPC in the micropig brain acutely following a central fluid percussion diffuse TBI[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This phenomenon; however, was not recapitulated following TBI in the rat[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], indicating that MPC onto injured axons might be species specific. The current findings that MPC onto injured axonal swellings occurs in human postmortem tissue, validates our previous findings in the micro pig and indicates that MPC following TBI might primarily manifest in higher order gyrencephalic brains. These findings show that MPC is a phenomenon that occurs in the human population, necessitating further investigation.\u003c/p\u003e \u003cp\u003eDiffuse axonal injury (DAI) or traumatic axonal injury (TAI) is one of the hallmark pathologies of mild TBI[\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. It was originally thought that DAI occurs following TBI due to the mechanical shearing of the axons. However, this is only true for a subset of axons that are observed to be injured within minutes of TBI, which is referred to as primary axotomy. Rather, secondary axotomy/axonal injury, which occurs sub-acutely after injury in rodent models of TBI is the phenomenon that is typically investigated. Secondary axonal injury involves accumulation of the cytoskeleton and organelles within the axons[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Specifically, the tensile forces of the TBI cause axonal alterations that allow an influx of calcium into the axon. This calcium influx leads to activation of cysteine protease pathways, which leads to degradation of the cytoskeleton and ultimately lead to a reactive axonal swelling as anterogradely transported proteins and organelles pool at the end of the proximal axon[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the early 1990s immunohistochemistry against the anterogradely transported protein, APP, was found to efficiently label the proximal injured axonal segment where it pooled[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Immunohistochemical labeling of APP has since become the gold standard for identifying DAI pathologically. Secondary axonal injury is typically studied in rodents 6\u0026ndash;24 hours following injury, when it is most prevalent[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Within higher order animals DAI appears to be prevalent starting hours following injury and peaking at 1w post-TBI[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], however DAI has been shown to last up to 6 months in a pig model of TBI[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and has been observed in human postmortem tissue from people several years following a TBI[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany recent studies have demonstrated the impact of inflammatory cascades in regulating behavioral morbidities, general pathology, and neuronal function in both the normal brain and in various disease states, including TBI[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Neuroinflammation has been demonstrated in various brain regions in the human population chronically following TBI[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Microglia, the innate immune cells of the brain, are critical mediators of these TBI-induced neuroinflammatory processes[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52 CR53 CR54 CR55 CR56\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Microglia have been shown to contact specific areas of the axon in the mouse brain during homeostasis including, the nodes of Ranvier[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], the axon initial segment[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], synapses[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], and neuronal soma[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Many studies using rodents have indicated that reduction or elimination of activated microglia and/or targeting various neuroinflammatory signaling pathways ameliorates downstream pathology and behavioral morbidity[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Conversely, other studies have also found that anti-inflammatory microglial activation is necessary and potentially advantageous[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. These studies demonstrated activated microglia can secrete neurotrophic factors[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e], which would suggest a potential ameliorative effect of microglia following injury in some cases. Additionally, recent studies have shown that microglia physical contacts play a role in regulating neuronal activity, either increasing activity following anesthesia[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e] or decreasing activity following epileptiform activity[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA study by Schirmer et. al. investigating human post-mortem tissue found a significant positive correlation between the density of microglia and axonal outgrowth as well as the duration of patient survival following TBI, however, they did not quantitatively investigate the physical interactions between microglia and injured axons[\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Another study done in vitro and in rats following a spinal cord injury found that exosomes from anti-inflammatory microglia increased neurite outgrowth in vitro and increased GAP43 expression in vivo, indicating that microglia could play a role in axonal outgrowth[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroglia have been observed within physical proximity to injured axonal swellings in human postmortem tissue[\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Oehmichen et al. observed an increase in CD-68\u0026thinsp;+\u0026thinsp;microglial cells areas of axonal injury in the white matter at least five days post-TBI in human postmortem tissue, however, they only observed limited physical interactions between the CD68\u0026thinsp;+\u0026thinsp;cells and the APP\u0026thinsp;+\u0026thinsp;axonal segments[\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Ryu et al. qualitatively identified areas in which Iba-1\u0026thinsp;+\u0026thinsp;microglia were in proximity to APP\u0026thinsp;+\u0026thinsp;injured axons in postmortem tissue from individuals following both motor vehicle accident and blast induced TBI[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Although, these previous studies indicated that there could be direct physical interactions between microglia and injured axonal segments, our current study is the first to quantitatively show that MPC occurs onto injured proximal axonal segments in human brain tissue following TBI.\u003c/p\u003e \u003cp\u003eWe do appreciate that there are limitations to the current study, mainly, that all cases were from male doners. There is evidence that males and females respond to TBI differently[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. A recent study also found that the burden of axonal injury following a diffuse TBI in a pig model was significantly higher in females compared to age matched males[\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Further, microglia have been shown to be different in males and females[\u003cspan additionalcitationids=\"CR82 CR83 CR84\" citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Therefore, investigations into MPC in both the male and female population should be done to fully appreciate the prevalence of MPC onto injury axons in the human brain. Additionally, these sections were only 15\u0026micro;m thick, precluding a 3D investigation of MPC, as was done in our previous micro pig studies[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is likely that the numbers of microglial processes we found converging onto the axonal segments in the current study were artificially lower than they might actually be due to the section thickness. Therefore, investigations using thicker tissue in which 3D reconstructions could be done, would be warranted to glean a better appreciation of the degree of MPC onto injured axonal swellings in human postmortem tissue of both males and females. Despite these limitations, the current study is the first to quantitatively demonstrate MPC onto injured axons in the human brain following TBI. These findings indicate that MPC is a component of human TBI and that further studies exploring the phenotypes and overall roles of the microglia involved in MPC following TBI should be investigated.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003eHuman brain samples were acquired from the Department of Defense (DoD)/Uniform Services University (USU) Tissue Repository. All cases were from males between 26\u0026ndash;69 years old (median age of 36 years) with a maximum postmortem interval of 1day. All identifiers were removed from the samples. Tissue was paraffin embedded and sectioned at 15um. Slides containing areas demonstrating axonal injury regardless of brain region were used for this study. A total of 11 human brain samples were used. Of these samples, 6 were confirmed TBI cases with demonstrated areas of axonal injury and 5 were controls. All study staff was blinded to case group throughout the labeling, imaging, and analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTo visualize the interactions between microglia and injured or intact axonal segments in the human brain multiplexed fluorescent immunohistochemistry was performed. To identify injured axons, an antibody against amyloid precursor protein (APP) was used, which indicates axonal transport issues indicative of axonal injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. To visualize microglia, an antibody against ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Intact, non-injured axonal fibers were visualized using an antibody against myelin basic protein (MBP).\u003c/p\u003e \u003cp\u003eIn this procedure, sections were deparaffinized by incubating slides in progressively more concentrated alcohols. Antigen retrieval was done by steaming the tissue in pH 6.0 citric acid buffer for 30min. Tissue was then blocked and permeabilized at room temperature in 5% normal goat serum (NGS), 2% bovine serum albumin (BSA) and 1.5% triton in phosphate buffered saline for 2hr followed by overnight incubation with a rabbit antibody against microglial Iba-1 (1:200; Cat.#019-19741 Wako; Richmond, VA, USA) at 4C\u0026deg; in 5% NGS/2% BSA/0.5% triton. Tissue was washed with 1%NGS/1%BSA in PBS at least six times prior to secondary antibody incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:700; Cat.# A- 11008, Life Technologies, Carlsbad, CA, USA) in 1%NGS/1%BSA/PBS at room temperature for 2hr. Tissue was washed in PBS at least four times prior to overnight incubation with a mouse antibody against the 22C11 clone of APP (1:200; Cat.#14-9749-82, ThermoFisher Scientific, Waltham, MA, USA) in 5% NGS/2% BSA/0.5% triton at 4C\u0026deg;. Tissue was washed with 1%NGS/1%BSA in PBS at least six times prior to the next secondary antibody incubation with Alexa Fluor 647-conjugated goat anti-mouse IgG (1:700; Cat.# A- 21237, Life Technologies, Carlsbad, CA, USA) in 1%NGS/1%BSA/PBS at room temperature for 2hr. Iba-1 and APP labeled tissue was washed in PBS at least four times prior to overnight incubation with a rat antibody against MBP (1:200; Novus) at 4C\u0026deg; in 5% NGS/2% BSA/0.5% triton. Tissue was washed with 1%NGS/1%BSA in PBS at least six times prior to the third secondary antibody incubation with Alexa Fluor 568-conjugated goat anti-rat IgG (1:700; Cat.# A- 11077, Life Technologies, Carlsbad, CA, USA) in 1%NGS/1%BSA/PBS at room temperature for 2hr. Tissue was washed in PBS at least four times. Multiplex labeled tissue was coversliped with Vectashield hard-set mounting medium with Dapi (Cat.#H-1500; Vector Laboratories, Burlingame, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMicroglial Process Convergence Analysis\u003c/h2\u003e \u003cp\u003eThe fluorescently immunolabeled slides were imaged on the Keyence BZ-X800 microscope (Keyence Corporation of America, Itasca, IL, USA) at 40X magnification. One section was analyzed for each case by an investigator blinded to group. A navigation super-image was generated using the far-red channel in which the APP\u0026thinsp;+\u0026thinsp;injured axonal swellings could be visualized. Images containing at least 1 APP\u0026thinsp;+\u0026thinsp;injured axonal swellings were captured or no APP\u0026thinsp;+\u0026thinsp;swellings but clean MBP labeling were captured. Fewer MBP only images were captured, as there were several analyzable MBP\u0026thinsp;+\u0026thinsp;intact myelinated fibers in each captured image, whereas there were few analyzable APP\u0026thinsp;+\u0026thinsp;axonal swellings in each captured image. At least 25 images were taken for most cases, however, only 13 images were captured for 1 case as no APP\u0026thinsp;+\u0026thinsp;swellings were identified. Across all samples a total of 161 non-injured axonal segments from controls, 36 non-injured axonal segments from injured samples, 105 injured axonal swellings from controls, and 173 injured axonal swellings from injured samples were analyzed for the current study. Fiji Image J software (National Institute of Health, Bethesda, MD, USA) was used to evaluate the 2D images. Image scales were set to 5.3 pixels/um\u003c/p\u003e \u003cp\u003eTo assess the interaction between microglia and injured axonal swellings, the APP\u0026thinsp;+\u0026thinsp;axonal swelling was traced using the freehand tool and measured for perimeter, area, shape descriptors (aspect ratio, circularity, round, solidity), integrated density, and mean grey value. The number of microglial processes and puncta that were directly touching the APP\u0026thinsp;+\u0026thinsp;axonal swelling was counted by hand. Then, the region encircling the APP\u0026thinsp;+\u0026thinsp;axonal swelling was enlarged by 5um and the microglial processes and puncta within the enlarged region was counted by hand.\u003c/p\u003e \u003cp\u003eIn order to visualize the interaction between microglia and intact axonal segments, a random number generator was used to generate x,y coordinates to choose a MBP\u0026thinsp;+\u0026thinsp;axonal segment on the image. The axonal segment was traced with the freehand tool and measured for perimeter, area, shape descriptors (aspect ratio, circularity, roundness, and solidity), integrated density, and mean grey value. The number of microglial processes and puncta that were directly touching the MBP\u0026thinsp;+\u0026thinsp;intact axonal segment was counted by hand. Then, the region encircling the axonal segment was enlarged by 5um and the number of microglial processes and puncta within the enlarged region were counted by hand.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe statistics were run using IBM SPSS software (IBM Corp., Armonk, NY). A Shapiro-Wilk test was conducted to test for normality of the data. As the data was not normally distributed, a Mann-Whitney U test was used to test differences between all APP\u0026thinsp;+\u0026thinsp;injured axonal swellings and all MBP\u0026thinsp;+\u0026thinsp;intact axonal segments. A Kruskal-Wallis test was run to assess differences across multiple groups. A Bonferroni post hoc was used to correct for multiple pairwise comparisons. Statistical significance was set to a \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05. Data is presented as means and standard error of the mean. All raw data is included in Supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work would not have been possible without the tissue from the Department of Defense (DoD)/Uniform Services University (USU) Tissue Repository and Neuropathology Core, which is funded by the Department of Defense and is housed within the Center for Neuroscience and Regenerative Medicine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eALW performed quantitative analysis and wrote the first draft of the manuscript. KG captured images for analysis and performed quantitative analysis. AL captured images for analysis, organized the data, edited the manuscript, managed the project, and secured funding for the completion of this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. Following publication these data will also be available on the Open Science Framework along with the analysis protocol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApproval for human experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study was reviewed by the Virginia Commonwealth University Institutional Review Board under IRB ID HM20029279 and was determined not to be research involving human subjects as defined by DHHS and FDA regulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by NINDS grants R21 NS126611 and R01 NS128104.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDewan MC, Rattani A, Gupta S, Baticulon RE, Hung Y-C, Punchak M, et al. Estimating the global incidence of traumatic brain injury. 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Journal of Neurotrauma. 2019;36:1040\u0026ndash;53.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microglial process convergence, Traumatic brain injury, Axonal injury, postmortem tissue","lastPublishedDoi":"10.21203/rs.3.rs-4713316/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4713316/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraumatic brain injury (TBI) affects millions globally, with a majority of TBI cases being classified as mild, in which diffuse pathologies prevail. Two of the pathological hallmarks of TBI are diffuse axonal injury and microglial activation. While progress has been made investigating the breadth of TBI-induced axonal injury and microglial changes in rodents, the neuroinflammatory progression and interaction between microglia and injured axons following brain injury in humans is less well understood. Our group previously investigated microglial process convergence (MPC), in which processes of non-phagocytic microglia directly contact injured proximal axonal segments, in rats and micropigs acutely following TBI. These studies demonstrated that MPC occurred on injured axons in the micropig, but not in the rat, following diffuse TBI. While it has been shown that microglia co-exist and interact with injured axons in humans post-TBI, the occurrence of MPC has not been quantitatively measured in the human brain. Therefore, in the current study we sought to validate our pig findings in human postmortem tissue. We investigated MPC onto injured axonal swellings and intact myelinated fibers in cases from individuals that sustained a TBI and control human brain tissue using multiplex immunofluorescent histochemistry. We found an increase in MPC onto injured axonal swellings, consistent with our previous findings in micropigs, indicating that MPC is a clinically relevant phenomenon that warrants further investigation.\u003c/p\u003e","manuscriptTitle":"Microglial process convergence onto injured axonal swellings, a human postmortem brain tissue study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 21:18:04","doi":"10.21203/rs.3.rs-4713316/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T07:31:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-26T01:07:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-23T21:49:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50022455818686708839883411283660911276","date":"2024-07-16T00:03:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8362902132908734597014513873878561452","date":"2024-07-12T18:21:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-12T12:38:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-12T07:11:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-12T03:33:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-10T09:32:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-09T15:53:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"03d7b2c0-9d05-4fae-8278-b4e9af7b8f05","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35257130,"name":"Biological sciences/Neuroscience/Glial biology/Microglia"},{"id":35257131,"name":"Health sciences/Neurology/Neurological disorders/Brain injuries"}],"tags":[],"updatedAt":"2024-09-16T16:06:33+00:00","versionOfRecord":{"articleIdentity":"rs-4713316","link":"https://doi.org/10.1038/s41598-024-71312-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-12 15:58:32","publishedOnDateReadable":"September 12th, 2024"},"versionCreatedAt":"2024-08-09 21:18:04","video":"","vorDoi":"10.1038/s41598-024-71312-7","vorDoiUrl":"https://doi.org/10.1038/s41598-024-71312-7","workflowStages":[]},"version":"v1","identity":"rs-4713316","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4713316","identity":"rs-4713316","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}