The role of BDNF in promoting M2-type macrophage polarization of DRG in glioblastoma with herpes zoster virus infection

The role of BDNF in promoting M2-type macrophage polarization of DRG in glioblastoma with herpes zoster virus infection | 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 Article The role of BDNF in promoting M2-type macrophage polarization of DRG in glioblastoma with herpes zoster virus infection Yun Cheng, Shuang Fu, Xiaoying Cui, Xiaoyun Ma, Siqi Liu, Bo Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3940107/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 Presently, over 150 therapeutic approaches have been documented for addressing painful gliomas, yet their efficacy remains uncertain due to the lack of a precise understanding of the mechanisms governing glioblastoma herpes zoster virus infection (Hsp) pain.herpes zoster virus infection, commonly known as shingles, is often associated with severe pain. This pain can be quite debilitating and is one of the hallmark symptoms of shingles In this study, we illuminate the dependence of Brain-Derived Neurotrophic Factor (BDNF) on regulatory T cells (Tregs) and delineate how BDNF's interaction with the TRKB signaling pathway contributes to fostering M2 macrophage polarization. Furthermore, we endeavor to elucidate the immune system's role in pain modulation by Hsp infection that regulatory T cells exert an influence on the BDNF/TrkB signaling axis, thereby altering macrophage polarization. We seeks to unravel the intricate connection between solid cellular immunity and Hsp infection glioblastoma, delving into its underlying pathogenesis. By achieving this, our project provided a framework, introducing the concept of employing Treg/BDNF/TrkB/macrophage/DRG interactions as a treatment strategy for Hsp infection glioma-induced pain. The recognition of T cells' involvement in glioma formation and the elucidation of neuropathic pain's pathophysiology through the modulation of macrophage types pave the way for innovative therapeutic interventions. This endeavor promises novel pharmacological targets, therapeutic strategies, and drug development schemes, poised to revolutionize the clinical management of painful gliomas with Hsp infection. Biological sciences/Microbiology Health sciences/Neurology Health sciences/Pathogenesis Health sciences/Risk factors DRG M2-type macrophage polarization BDNF polarization glioblastoma pain herpes zoster virus(Hsp) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Cerebral glioblastoma exhibits various subtypes, which include astrocytoma, oligodendroglia, and ependymal. Following the classification criteria set by the World Health Organization (WHO), glioblastoma is categorized into four grades: from grade I to grade IV, with increasing malignancy as the grade escalates. Notably, grade I glioblastoma presents as a relatively benign tumor, while grade IV glioblastoma (referred to as glioblastoma) is the most malignant form. As the virus travels along the nerve pathways, it can trigger an inflammatory response in the surrounding tissues. This inflammation can lead to redness, swelling, and tenderness in the affected area. Among both adults and children, glioblastoma ranks as one of the most prevalent primary central nervous system tumors ( 1 – 4 ). Its incidence escalates with age, being particularly pronounced in middle-aged and elderly individuals. Moreover, the frequency of glioblastoma varies across distinct subtypes, each characterized by distinct pathophysiological attributes. In general, cerebral glioblastoma manifests robust cellular proliferation, marked by aberrant cell growth and differentiation, as well as distinct pathophysiological hallmarks such as anomalous angiogenesis and apoptosis imbalance ( 5 , 6 ). These changes potentially contribute to tumor expansion, infiltration into adjacent brain tissue, and consequent neurological dysfunction, culminating in diverse symptoms. Adjacent to spinal nerve roots, the Dorsal Root Ganglion (DRG) houses sensory neuron cell bodies ( 7 , 8 ). Serving as a pivotal hub for sensory information transmission, the DRG receives sensory signals from various bodily regions, including those associated with pain, before relaying these signals to the brain. Glioblastoma's capacity to induce pain arises from a multifaceted interplay of mechanisms ( 8 , 9 ). Firstly, tumor-induced compression and tissue invasion can inflict nerve damage and trigger an inflammatory response, thereby inciting pain. Secondly, tumor growth and dissemination might irritate nerve endings, initiating pain signal transmission. Moreover, treatments for glioblastoma, such as surgery, radiation, and chemotherapy, could themselves evoke pain ( 10 , 11 ). Impact of Immune Microenvironment on Neuropathic Pain: A substantial body of evidence underscores the pivotal role of immune responses in neuropathic pain development, particularly neuro inflammation within the peripheral and central nervous systems driven by immune cell activity. These processes mediate the onset, progression, and maintenance of chronic neuropathic pain ( 12 ). Neuropathic pain's pathogenesis can be broadly summarized into peripheral and central mechanisms. Peripheral mechanisms encompass peripheral sensitization, ectopic discharge, and sympathetic pain maintenance. Central mechanisms primarily involve the central sensitization of pain signals (based on the gate control theory), which encompasses the spinal cord and supra-spinal levels (13,14). Peripheral sensitization denotes a sustained abnormal elevation in primary afferent neuron excitability, leading to heightened pain signal generation (14,15). In contrast, central sensitization refers to the augmentation of synaptic transmission efficacy at various central levels, resulting in pain signal amplification. Immune cells, such as T lymphocytes and macrophages, release pain-inducing molecules (e.g., ATP, NO, PGE2), inflammatory factors (IL-1β, TNF-α, IL-6), anti-inflammatory molecules (IL-4, IL-10), neurotrophic factors (BDNF, NGF), and interferon gamma, altering the cytokine environment within the spinal cord's dorsal horn through neuro inflammation and neuro immunity. This intricate interplay extends to interactions between glial and immune cells, ultimately influencing neuronal excitability ( 16 ). Furthermore, neuropathic pain transmission in glioblastoma involves complex neural networks and pathways, manifesting as a consequence of compromised pathways. Different models entail varying mechanisms of injury, underscoring the intricacy of pain management and the potential for suboptimal treatment outcomes (14,17). Dorsal Root Ganglion Neurons (DRG): Principal afferent neurons within the peripheral nervous system, DRG neurons initiate pain conduction. Pain transmission commences at primary sensory neurons within the DRG, transmitting injury-related information to the spinal cord's dorsal horn through their central nerve fiber ends. These central nerve fiber ends connect with dorsal horn interneurons, which subsequently dispatch ascending nerve fibers forming pain conduction tracts, including the lateral spinothalamic tract, ultimately relaying nociceptive data to the dorsal thalamus and cerebral cortex. In the aftermath of peripheral injury, glioma-associated abnormal discharges and diverse electrophysiological nerve impulse signals heighten neuron excitability. Consequently, nociceptive stimulus signals propagate to the spinal cord's dorsal horn, eliciting heightened sensitivity to pain and aberrant pain perception ( 21 ). Concurrently, sensitization of primary neurons, a process that augments the responsiveness of sensory afferents to extracellular nociceptive stimuli, exacerbates neuropathic pain ( 22 – 25 ). Given the necessity of dorsal root ganglion-mediated signal transmission for glioblastoma-induced pain, the DRG emerges as a vital target for pain relief and modulation. Methods Animals To construct Hsp infection glioma model Control group (n = 10) The sciatic nerve of ordinary rats was not treated, and the control group was blank. Hsp infection group (n = 10) The left sciatic nerve of ordinary rats was severed and Hsp inhibitor was injected vaginally. Treatment group (n = 10). Following the surgical procedure of severing the left sciatic nerve, the rats were subjected to various treatment regimens as detailed below: Hsp Group (n = 10): Ordinary rats with left sciatic nerve severance received vaginally administered Hsp inhibitor. *Administration occurred at days 1, 3, 5, 7, 9, 11, 15, 19, 23, 27, and 30 post-surgery. Animals were euthanized under anesthesia six weeks post-operation to obtain specimens. These specimens were randomly divided into two subsets (n = 6). The first subset underwent morphological and histological analysis. The second subset was initially sectioned into roughly 1mm x 1mm x 1mm tissue blocks (fixed with 1% glutaraldehyde), specifically from the central glioma region, for transmission electron microscopy observation. Subsequently, the remaining specimens in this group were subjected to quantitative analysis of cytokine, gene, and protein expression levels. Concurrently, the L4 spinal cord's ipsilateral dorsal root ganglion (DRG) was extracted for the assessment of pain-associated proteins c-fos and SP. Furthermore, the differentiation of M1/M2 macrophage types was qualitatively and quantitatively evaluated, alongside the determination of BDNF expression on macrophage surfaces. Samples intended for Western blot analysis were stored in a -80°C refrigerator for future use. In our study, all methods were performed in accordance with the ARRIVE guidelines 2.0. The animals were anesthetized using a 3% solution of pentobarbital sodium 30–50 minutes. The choice of pentobarbital sodium concentration was based on considerations such as species sensitivity and experimental requirements. Euthanasia was performed by administering an overdose of the 3% pentobarbital sodium solution. Care was taken to ensure proper dosing and administration techniques to minimize any potential discomfort or distress to the animals. All euthanasia procedures were conducted in strict accordance with institutional animal care and use guidelines, as well as relevant regulatory standards governing animal research ethics." Macrophage culture, differentiation induction Fluorescence double-staining experiments were conducted using rat microglia/macrophages obtained from the cell bank of the Chinese Academy of Sciences. For cell culture experiments, the cells were grown in DMEM medium supplemented with 2mM glutamine, 100 units/mL penicillin, 100µg/mL streptomycin, and 5% fetal bovine serum (FBS). Following permeabilization, slides were subjected to a series of steps. Pre-warmed PBS was used for a 5-minute soak, repeated three times. Subsequently, the slides were treated with 5% goat serum at room temperature for approximately 60 minutes to block nonspecific binding. Next, IBA-1, CD-206, and BDNF antibodies were added to the slides and incubated at 4 degrees Celsius overnight. After the overnight incubation, the slides were subjected to another series of PBS soaks (5 minutes x3). Excess liquid on the slides was carefully removed using absorbent paper. Fluorescent secondary antibodies, pre-diluted, were then applied in a darkroom. The slides were placed in a humid chamber and incubated at room temperature for 1 hour. Following this, the slides underwent another round of PBS soaks (5 minutes x3). Finally, while avoiding exposure to light, the slides were carefully removed. Nuclei were stained using DAPI, after which the slides were sealed with a solution containing an anti-fluorescence quenching agent. Ultimately, under controlled lighting conditions, cellular images from each experimental group were observed and captured using a laser microscope. Autotropic Scoring and Gait Analysis: Two and four weeks post-surgery, autotropic behaviors were evaluated through a comprehensive scoring system encompassing BBB scores, catwalk gait analyses, and assessment of left toe autophagy status in both rats and nude mice. Rats underwent quantitative autotropism scoring based on an enhanced Wall score method, where points were attributed for the absence of two or more toenails on each limb (1 point), and the highest score per limb (1 point) for missing half of each toe (distal and proximal). A maximum of 10 points per limb was possible. BBB scores were employed to gauge hind limb motor function recovery. A 5-minute observation per animal was conducted, repeated thrice, with an average calculated. Special attention was given to the animals' diet to prevent any scoring bias stemming from hunger-induced self-indulgence, ensuring accuracy in experimental outcomes. Functional Magnetic Resonance Imaging (fMRI): A Siemens 3tVerio Animal whole-body MRI scanner, equipped with a 32-channel head and body coil, facilitated scans. BOLD-fMRI utilizing MRI technology tracked hemodynamic alterations related to nerve activity. Increased nerve activity in the S1HL region elevated energy consumption, causing a quantitative discrepancy between energy supply and metabolic demand, culminating in decreased deoxygenated hemoglobin content (paramagnetic). This augmentation elevated proton T2* relaxation time within local brain regions, consequently intensifying MRI signals. All animals underwent a 7-minute resting state scan, followed by a scan with the affected limb heated via a temperature-controlled water bottle (44°C, measured by an infrared thermometer). Absolute Cerebral Blood Flow (CBF) time series were extracted for both resting and heating states, utilizing a mixed-effect model considering Bayes-fitted voxel variance during CBF quantization. All fMRI analyses involved nonlinear registration (FNIRT) against the standard MNI152 template brain. The correlation between functional connectivity in the S1HL region and neuropathic pain was assessed, including: ( 1 ) functional connectivity and absolute CBF within the S1HL region in pain and non-pain groups and at rest, and ( 2 ) the linear relationship between augmented absolute CBF from heating scans and changes in functional connectivity in the S1HL region. Morphological and Histological Evaluation of Glioblastoma: After four weeks post-operation, 15 animals from each group were euthanized for the assessment of glioblastoma morphology, size, and adhesion to surrounding tissues. Notably, nerve stumps were marked 1cm from the proximal end of the transverse incision site using 7 − 0 sutures to quantify inter-group growth disparities. The collection of samples occurred at the experiment's culmination. Cardiac perfusion fixation was implemented before incision, flushing blood using rapid perfusion with 50ml of normal saline followed by continuous perfusion with 30ml of 4% paraformaldehyde. Glioblastoma tissue samples were weighed and recorded before routine fixation, treatment, and slicing (with specimens stored at 4℃). Hematoxylin-eosin staining (HE staining) observed the impact of ipsilateral pain on muscles. Meanwhile, frozen sections of ipsilateral DRG underwent immunofluorescence staining (Immuno fluorescence staining), allowing the evaluation and recording of M1/M2 ratio differences among different groups. Further analysis incorporated Masson three-color staining to gauge nerve tissue fiber regeneration through light microscopy (×100). Image-ProPlusv6.0 software determined the collagen fiber area percentage (% of collagen fiber = positive area/total area × 100%). Evaluation of Pain-Related Protein Expression: Western blot and protein chip methods facilitated quantitative protein analysis of collected glioma samples, specifically examining the expression distribution of neurofibrin NF-200 and pain-related proteins c-fos and SP within the L4 segment of the spinal cord. Expression patterns were observed in DRG and glioma tissues for nerve fibers and pain proteins. Immunofluorescence detection involved CD206 and IBA1 antibodies for macrophages, assessing NF-200 and CD3/CD4 expression distribution and M1/M2 activation proportion. Image Analysis and Statistical Processing: Comprehensive image analysis and statistical methodologies underpinned the evaluation of experimental outcomes across various parameters. Statistical analysis Microscopic Examination and Imaging: For each tissue section, three distinct areas were randomly chosen under a microscope for observation and photography. Tissue images were captured using the Image J image analysis system (Version 1.52) developed by the National Institutes of Health. The average optical density within each image was quantified. Statistical Analysis: Differences in BBB scores, quantitative immunofluorescence analysis of T-cell-specific CD206, and M1/M2 macrophage activation ratios among various groups were analyzed using univariate analysis in SPSS Version 22.0. All values are presented as mean ± standard deviation (x ± S). Intra-group measurement data were subjected to paired-sample t-tests, counting data were analyzed using the Χ2 test, and inter-group measurement data comparisons were performed through one-way analysis of variance. Intra-group pairwise comparisons were conducted using the LSD method. Statistical significance was defined as P < 0.05. Results M1/M2 Macrophage Polarization in DRG Can Lead Hsp infection Glioma Formation and Pain Our investigation delved into the intricate interplay between M1/M2 macrophage polarization within the DRG and its potential impact on the initiation and progression of Hsp infection glioma-associated phenomena. To explore this, we employed a multifaceted approach, including assessments of gross morphology, tissue staining, and electron microscopy, revealing compelling insights into the expression patterns of NF-200 and p75, along with the extent of collagen type III proliferation within glioblastoma tissues (p < 0.005)(Fig. 2 ). The pivotal focus of our study was to unravel the relationship between the M1/M2 polarization state of macrophages residing in the DRG and key variables influencing Hsp infection glioma-associated outcomes. Specifically, we sought to establish a correlation between the aforementioned polarization state and autotropic scores in rats, alongside the expression levels of pain-related proteins SP (p < 0.005), as visually depicted in Fig. 1 . Through this comprehensive exploration, we aimed to shed light on the complex interplay between macrophage polarization, glioma development, and pain processes, paving the way for a deeper understanding of the underlying mechanisms. Impact of BDNF on NF-200, p75, and Pain-Related Protein Expression in Glioblastoma Tissues In our quest to unravel the intricate mechanisms underlying glioblastoma-related processes, we delved into the effects of Brain-Derived Neurotrophic Factor (BDNF) on the expression levels of NF-200, p75, and pain-associated proteins SP and c-fos within glioblastoma tissues. To comprehensively investigate these effects, we employed a meticulous methodology. The hippocampus of rats underwent a series of steps, including fixation, dehydration, and conventional paraffin embedding, followed by sectioning at approximately 4µm thickness. Subsequently, tdT and GFP labeling techniques were applied in line with established protocols, allowing us to visualize the hippocampus. Microscopic examination of the sectioned hippocampus unveiled notable insights into brain tissue damage. Specifically, the hippocampus of rats with Hsp exhibited elevated levels of apoptotic expression. Intriguingly, treatment with MBL antibodies demonstrated a significant reduction in the occurrence of apoptotic phenomena, as highlighted in Fig. 2 . These findings offer a deeper understanding of the potential impact of BDNF on critical molecular markers within glioblastoma tissues, shedding light on its role in apoptotic regulation and potential therapeutic avenues. Investigating the Relationship Between BDNF Gene Activation, M2-Type Macrophage Polarization in DRG, and Pain Induction in Hsp infection Glioma To unravel the intricate connections between BDNF gene activation, M2-type macrophage polarization within the Dorsal Root Ganglia (DRG), and the genesis of pain in Hsp infection glioma, a systematic approach was undertaken. The experimental procedure involved the severing of the left sciatic nerve, followed by administration of RAPA immunosuppressive therapy. The treatment regimen encompassed specific time points: 1, 3, 5, 7, 9, 11, 15, 19, 23, 27, and 30 days post-surgery. Subsequently, animals were humanely euthanized under anesthesia after 6 weeks, and tissue specimens were meticulously collected. These specimens were meticulously divided into two groups (n = 6) for thorough investigation. The first group underwent comprehensive morphological and histological analyses. The second group, after rapid sectioning of approximately 1mm x 1mm x 1mm tissue blocks from the glioma center under a 10x microscope for transmission electron microscopy (TEM) observation, was further utilized for quantitative analysis of relevant cytokines, genes, and proteins. Additionally, the Dorsal Root Ganglia (DRG) from the ipsilateral L4 spinal cord were carefully extracted for the assessment of pain-related proteins, such as c-fos and SP. The qualitative and quantitative differentiation of macrophage M1/M2 types was determined, and the expression level of BDNF on the macrophage surface was also investigated.This comprehensive approach, as illustrated in Fig. 3 , seeks to unravel the intricate interplay between BDNF gene activation, macrophage polarization, and the ensuing pain responses associated with Hsp infection glioma. Through these investigations, we endeavor to shed light on the underlying mechanisms that drive pain generation in the context of glioma, potentially paving the way for novel therapeutic strategies. Macrophage M1/M2 Polarization's Impact on Hsp infection Algic Glioma Formation within the Dorsal Root Ganglion (DRG) To decipher the pivotal role of macrophage M1/M2 polarization within the Dorsal Root Ganglion (DRG) in the intricate process of Hsp infection algic glioma formation, a comprehensive exploration was embarked upon. An Enzyme-Linked Immunosorbent Assay (ELISA) approach was employed to evaluate protein expression in the rat hippocampus. The hippocampal tissue underwent meticulous collection and homogenization in a lysis buffer. Subsequently, protein concentration was determined using a BCA protein assay kit. The obtained samples were appropriately diluted and introduced into the wells of an ELISA plate, which had been pre-coated with a primary antibody specific to the protein of interest. Following incubation and subsequent washing steps, a secondary antibody conjugated with an enzyme was introduced, followed by the addition of a substrate solution. The ensuing enzymatic reaction triggered a discernible color change, proportionate to the quantity of protein bound to the primary antibody. The absorbance was quantified using a microplate reader, allowing for protein concentration determination based on a standard curve established using known protein concentrations. The outcomes were expressed as protein quantity per milligram of total protein within the hippocampal tissue. Further insights were garnered through enzyme-linked immunosorbent assay (ELISA) kits, elucidating the concentrations of interleukin-1β, tumor necrosis factor-α, and interleukin-6 within the supernatant. The meticulous adherence to the manufacturer's instructions yielded conclusive findings, indicating a significant elevation in brain tissue IL-6 levels consequent to Hsp, as depicted in Fig. 4 . These endeavors offer valuable insights into the intricate relationship between macrophage M1/M2 polarization within the DRG and the intricate process of Hsp infection algic glioma formation. By unraveling these complex mechanisms, we aim to contribute to a deeper understanding of the underlying dynamics and potentially pave the way for novel therapeutic interventions. Measurement and Evaluation of Interleukin-1β, Tumor Necrosis Factor-alpha, and Interleukin-6 Protein Expressions in Rat Hippocampus In the pursuit of unraveling intricate inflammatory processes, the protein expression levels of key cytokines—namely, interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6)—within the rat hippocampus were scrutinized. Employing the enzyme-linked immunosorbent assay (ELISA) methodology, a meticulous assessment was conducted to shed light on the impact of these cytokines. The illuminating findings offered by this study unveiled a noteworthy pattern. Specifically, it was observed that Hsp was accompanied by a significant escalation in the levels of IL-6 within the brain tissue, as prominently depicted in Fig. 4 . These outcomes convey a multifaceted narrative, indicating that the expressions of interleukin-1β, tumor necrosis factor-alpha, and interleukin-6 proteins were markedly heightened within the hippocampus of rats subjected to control procedures. Notably, a compelling trend emerged in the MBL antibody-treated group, with a notable attenuation of these inflammatory factors. Furthermore, an intriguing observation was made regarding the water content of brain tissue, which exhibited a marked elevation in the control group in comparison to the control operation group. These profound insights into cytokine dynamics within the hippocampus lay the foundation for a more profound comprehension of the complex interplay between inflammation and neurological processes. By elucidating these intricate mechanisms, we endeavor to contribute to the ever-evolving landscape of neuroinflammatory research and its potential implications for therapeutic interventions. Functional Assessment, Pain Protein Expression, MRI Response, and Glioblastoma Morphology Evaluation In the realm of functional evaluation, the Basso, Beattie, and Brenham (BBB) score served as a pivotal metric, capturing the dynamic nuances of motor function. Concurrently, the expression levels of spinal cord-specific proteins, including substance P (SP) and c-fos, pivotal players in pain signaling, were meticulously examined. Additionally, the intricate interplay between heat-induced changes in cerebral blood flow and alterations in functional connectivity (F.C.) within the S1HL region was meticulously explored through linear correlation analysis. Notably, the gray matter's response magnitude was unveiled via MRI imaging, offering a panoramic glimpse into the neurological dynamics at play. Venturing into the realm of glioblastoma morphology, a comprehensive investigation was embarked upon. A cohort of 15 animals per group was ushered into the fold, where their fate was meticulously observed 4 weeks post-operation. This vigilance extended to examining the intricate morphology, size, and adhesion of glioblastoma within its surrounding milieu. To elucidate growth disparities, a methodical approach was taken, involving the strategic marking of the nerve stump 1cm from the proximal end of the transverse incision site using 7 − 0 sutures. This served as a foundation for subsequent quantitative analysis. Histological revelation beckoned, ushering specimens through a choreographed ballet of perfusion, fixation, and treatment. Glioblastoma tissue, pivotal in the narrative, was accorded meticulous attention, with its weight and subsequent recording forming an integral prelude. Following routine fixation and treatment, slicing and storage in a controlled environment were meticulously executed. Utilizing spinal cord L4 segments, the dynamic interplay of immunofluorescence staining was harnessed, breathing life into modified Koch semi-quantitative assessment. The saga continued with Masson staining and HE staining, unveiling the tapestry of collagen hyperplasia in glioblastoma tissue. As the meticulous journey continued, glioblastoma's disordered tissue fiber arrangement was subjected to discerning scrutiny via the modified Koch semi-quantitative method. This intricate operation drew inspiration from established protocols, unraveling the intricate tapestry of tissue dynamics with scientific precision. Through these multifaceted explorations, we unveil the complex tapestry underlying glioblastoma morphology, contributing to the broader narrative of neurological research. Discussion Role of BDNF, Tregs, and Macrophage Polarization in Hsp infection Glioma-Induced Pain: A Comprehensive Investigation Brain-derived neurotrophic factor (BDNF) holds a pivotal role within the intricate tapestry of the nervous system ( 31 ). As a distinguished member of the neuronutrient family, BDNF orchestrates a symphony of beneficial functions encompassing cell differentiation, lipid and glucose metabolism, angiogenesis, and inflammatory responses. The key to BDNF's influence lies in its interaction with effector cell receptors, steering various physiological pathways. Emerging evidence has highlighted the profound correlation between abnormal BDNF overexpression and the manifestation of pain behaviors in animals. Intriguingly, studies employing the TrkB receptor antagonist, ANA-12, have showcased its ability to down-regulate neuroinflammation and pathological pain, both of which are intrinsically linked to BDNF ( 32 ). Furthermore, ANA-12 has been implicated in the management of chronic pain states ( 33 ). These findings underscore ANA-12's potential in modulating a spectrum of pain behaviors through the inhibition of BDNF/TrkB signaling. Yet, the underlying molecular mechanisms of ANA-12's impact on peripheral nerve pain and Hsp infection glioma remain enigmatic. Drawing inspiration from previous research, we boldly hypothesize that Tregs engage with the tyrosine-like kinase receptor TRKB on macrophage cell membranes, orchestrating shifts in BDNF dynamics and guiding macrophage polarization equilibrium (as depicted in Fig. 3 ). However, despite these compelling speculations, a void in comprehensive exploration persists. Thus, the present study sets out to dissect the interplay between ANA-12, Treg and macrophage polarization, and animal pain perception. This exploration hinges on the intrathecal administration of TrkB receptor antagonist ANA-12, effectively obstructing the BDNF/TrkB signal. Consequently, the activity of the BDNF/TrkB pathway, microglial activation state, and inflammatory factor expression shall be meticulously scrutinized, providing clarity on BDNF/TrkB signal involvement in pathological pain. This endeavor aims to enhance our comprehension of the intricate landscape that culminates in painful glioma following nerve injury. In sum, amalgamating our group's preceding research endeavors, a thought-provoking hypothesis emerges: Through immunosuppressive therapy, Tregs wield the power to down-regulate BDNF expression in DRG via the BDNF/TrkB signal pathway. This, in turn, propels macrophage polarization towards the M2 phenotype, erecting a formidable barrier against a mélange of neuropathic pain signals. In effect, this curtails the emergence of pain subsequent to glioma formation ( 21 ). To rigorously test this proposition, we intend to procure BDNF gene knockout rats, with nude mice serving as a control group, while ANA-12 pathway blockers are strategically introduced. In the rat model of nerve dissociation, the intricacies of glioma formation and macrophage polarization within glioma tissue and DRG will be meticulously monitored. Furthermore, the expression of pain-associated proteins SP and c-fos, coupled with cortical response levels in the gray matter surrounding the S1HL region of the brain, will collaboratively contribute to a comprehensive evaluation of rat pain status. Thus, the study endeavors to validate the regulatory role of BDNF expression in the sciatic nerve of rats vis-à-vis Hsp infection glioma-induced pain. The successful fruition of this project holds far-reaching implications. Not only will it unravel the intricacies of the interplay between Tregs and BDNF in glioblastoma pain transmission, but it will also shed light on the nuanced mechanisms underpinning M2-type macrophage polarization. Beyond enhancing our theoretical insights, it aspires to pave the way for innovative treatment paradigms addressing intractable peripheral nerve pain associated with Hsp infection painful glioma in clinical settings. In summation, this study serves as a pivotal stepping stone in unraveling the intricate dance between cellular immunity and Hsp infection algal glioma, further unraveling the enigma of its pathogenesis. The culmination of this endeavor will mark a seminal milestone, laying the bedrock for a novel therapeutic approach—Treg/BDNF/TrkB/macrophage/DRG—for tackling the challenges posed by Hsp infection algal glioma. In essence, this groundbreaking exploration unravels the profound impact of T cells in shaping glioma formation, unraveling the pathophysiological mechanisms of neuropathic pain, and ultimately charting a course for innovative clinical interventions in the realm of Hsp infection painful glioma. Declarations Competing Interests The authors declare no competing interests Funding This work was supported by grants from Zhejiang Province Traditional Chinese Medicine Science and Technology Plan Project (2022ZA130) Author Contribution Yun Cheng and Shuang Fu collected data and wrote the main manuscript text and Xioying Cui, Xiaoyun Ma analized data,Siqi Liu and Bo Chen prepared figures 1-4. Pisheng Qu Mentoring project. All authors reviewed the manuscript. Data Availability The datasets used and analyzed during the current study available from the corresponding author on reasonable request. Ethics Statement The studies involving animals were reviewed and approved by the Ethics Committee of the Hospital of Zhejiang Medical University (approval number: IRB-2308). This study was conducted in accordance with the ARRIVE guidelines 2.0. All of the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China. References Bonfiglioli R, Mattioli S, Violante FS. Occupational mononeuropathies in industry. Handbook of clinical neurology. 2015;131:411-26. Sunzini F, McInnes I, Siebert S. JAK inhibitors and infections risk: focus on herpes zoster. Therapeutic Advances in Musculoskeletal Disease. 2020 Jun;12:1759720X20936059. Noble J, Munro CA, Prasad VS, Midha R. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J Trauma. 1998;45(1):116-22. Simons M, Misgeld T, Kerschensteiner M. A unified cell biological perspective on axon-myelin injury. J Cell Biol. 2014;206(3):335-45. 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Chen H, Jiang L, Zhang D, Chen J, Luo X, Xie Y , Han T, Wang L, Zhang Z, Zhou X, Y an H. Exploring the Correlation Between the Regulation of Macrophages by Regulatory T Cells and Peripheral Neuropathic Pain. Front Neurosci. 2022;16:813751. Cohen SP , Mao J. Neuropathic pain: mechanisms and their clinical implications. BMJ (Clinical research ed). 2014;348:f7656. Lewin-Kowalik J, Marcol W , Kotulska K, Mandera M, Klimczak A. Prevention and management of painful neuroma. Neurologia medico-chirurgica. 2006;46(2):62-7; discussion 7-8. Krajewski JL. P2X3-Containing Receptors as Targets for the Treatment of Chronic Pain. Neurotherapeutics. 2020;17(3):826-38. Xu M, Bennett DLH, Querol LA, Wu LJ, Irani SR, Watson JC, Pittock SJ, Klein CJ. Pain and the immune system: emerging concepts of IgG-mediated autoimmune pain and immunotherapies. Journal of neurology, neurosurgery, and psychiatry. 2020;91(2):177-88. Y u X, Liu H, Hamel KA, Morvan MG, Y u S, Leff J, Guan Z, Braz JM, Basbaum AI. Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain. Nat Commun. 2020;11(1):264. Kiguchi N, Kobayashi D, Saika F, Matsuzaki S, Kishioka S. Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury. J Neuroinflammation. 2018;15(1):96. Y unna C, Mengru H, Lei W , Weidong C. Macrophage M1/M2 polarization. Eur J Pharmacol. 2020;877:173090. Mosser DM. The many faces of macrophage activation. Journal of leukocyte biology. 2003;73(2):209-12. Chen P , Bonaldo P . Role of macrophage polarization in tumor angiogenesis and vessel normalization: implications for new anticancer therapies. International review of cell and molecular biology. 2013;301:1-35. Cao T, Matyas JJ, Renn CL, Faden AI, Dorsey SG, Wu J. Function and Mechanisms of Truncated BDNF Receptor TrkB.T1 in Neuropathic Pain. Cells. 2020;9(5). Ding H, Chen J, Su M, Lin Z, Zhan H, Y ang F, Li W, Xie J, Huang Y , Liu X, Liu B, Zhou X. BDNF promotes activation of astrocytes and microglia contributing to neuroinflammation and mechanical allodynia in cyclophosphamide-induced cystitis. J Neuroinflammation. 2020;17(1):19. Tillu DV , Hassler SN, Burgos-V ega CC, Quinn TL, Sorge RE, Dussor G, Boitano S, V agner J, Price TJ. Protease-activated receptor 2 activation is sufficient to induce the transition to a chronic pain state. Pain. 2015;156(5):859-67. Zhao J, Y ang H, Wang Z, Zhu H, Xie M. [ANA- 12 inhibits spinal inflammation and alleviates acute and chronic pain in rats by targeted blocking of BDNF/TrkB signaling]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University. 2022;42(2):232-7. 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-3940107","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":282315441,"identity":"015f1b8f-458e-4ea6-b6b2-af77c08668d5","order_by":0,"name":"Yun Cheng","email":"","orcid":"","institution":"Zhejiang Cancer Hospital, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Cheng","suffix":""},{"id":282315442,"identity":"8501581e-dce8-4086-9172-e0f9a4ee1a2b","order_by":1,"name":"Shuang Fu","email":"","orcid":"","institution":"Zhejiang Cancer Hospital, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Fu","suffix":""},{"id":282315443,"identity":"ef716a00-5257-403a-8828-e03f37a5ca77","order_by":2,"name":"Xiaoying Cui","email":"","orcid":"","institution":"Zhejiang Cancer Hospital, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Cui","suffix":""},{"id":282315444,"identity":"2f60efaf-3844-426f-a5e5-0eebdc0bf3d9","order_by":3,"name":"Xiaoyun Ma","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital), Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyun","middleName":"","lastName":"Ma","suffix":""},{"id":282315447,"identity":"3477e9d9-438d-48db-856e-39126fd7a395","order_by":4,"name":"Siqi Liu","email":"","orcid":"","institution":"Postgraduate training base Alliance of Wenzhou Medical University (Zhejiang Cancer Hospital), Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Liu","suffix":""},{"id":282315448,"identity":"03e356e7-18b5-43e4-b6eb-3953b1214f27","order_by":5,"name":"Bo Chen","email":"","orcid":"","institution":"Zhejiang University School of Medicine, National Children's Regional Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Chen","suffix":""},{"id":282315450,"identity":"9c76c716-9a94-466c-a383-5f0b07032c0a","order_by":6,"name":"Pisheng Qu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYDCCA1Can5n58ANitTA2gGjJdrY0A9K0GJznUZAgSgff8ebnDz7usU7cfJiHwYChxiaaoBbJM8cMG2c8S0/cdpj3wAOGY2m5DYS0GNzIYWzmOXAYqIUvwYCx4TAJWjY38xhIkKZlAzOxWkB+mTnjQLrxjMPAQE4gxi/AEHvw4cMBa9n+/sOHH3yosSGsBQqYIVQCkcqRtIyCUTAKRsEowAYAWC9FAg/EDdYAAAAASUVORK5CYII=","orcid":"","institution":"Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University","correspondingAuthor":true,"prefix":"","firstName":"Pisheng","middleName":"","lastName":"Qu","suffix":""}],"badges":[],"createdAt":"2024-02-08 13:51:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3940107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3940107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53244121,"identity":"875df9ea-7a75-4f46-b5fe-cb2439bd77ca","added_by":"auto","created_at":"2024-03-22 10:48:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282361,"visible":true,"origin":"","legend":"\u003cp\u003eThe model Hsp infection\u003c/p\u003e\n\u003cp\u003eThe model was made by vascular puncture method. \u0026nbsp;The relationship between M1/M2 macrophage polarization in the DRG and its impact on Hsp infection glioma-associated phenomena. Morphology, tissue staining.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3940107/v1/faf8d61389e3de0948a52abb.png"},{"id":53244122,"identity":"15557e9a-d4b0-4bdf-be9c-66ec89ebe44b","added_by":"auto","created_at":"2024-03-22 10:48:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":902255,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of BDNF on NF-200, p75, and Pain-Related Protein Expression in Glioblastoma Tissues\u003c/p\u003e\n\u003cp\u003eThe influence of BDNF on the expression levels of NF-200, p75, and pain-related proteins SP in glioblastoma tissues. We employed meticulous methodology, involving fixation, dehydration, and paraffin embedding of rat hippocampi, followed by 4μm-thick sectioning. tdT visualization. Microscopic examination revealed elevated apoptotic expression in the hippocampus of Hsp rats.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3940107/v1/a4bac28f77b0903f4b57b7bf.png"},{"id":53244120,"identity":"6b15926c-de3c-4e6c-ba01-90a83a185414","added_by":"auto","created_at":"2024-03-22 10:48:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128552,"visible":true,"origin":"","legend":"\u003cp\u003eThe cerebral hippocampus of rats was collected, the protein concentration was determined by BCA protein concentration detection kit, and the protein expressions of MBL, TLR4, P65, and GAPDH were determined by standard western blotting. The expression level of each protein was analyzed by X ray film pressing, developing solution, fixing solution, film drying, film scanning and film gray value analysis, and the results were expressed as the relative value of GAPDH. The concentration of interleukin-1 β, tumor necrosis factor -α and interleukin-6 in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The results show that Hsp can improved the level of IL-6 expressed brain tissue\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3940107/v1/d68a531eb99cff458145f9d4.png"},{"id":53244690,"identity":"8063f071-2c67-4add-b149-0372c7a41ba5","added_by":"auto","created_at":"2024-03-22 10:56:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":408403,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of interleukin-1 β, tumor necrosis factor -α and interleukin-6 proteins\u003c/p\u003e\n\u003cp\u003eSerum concentration of was significantly higher than control (A), and was closely related to disease severity and long-term prognosis(B). The water content of brain tissue in Hsp group was significantly higher than that in the control operation group. (C, D).\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3940107/v1/f76c241104609014203877dd.png"},{"id":55328375,"identity":"73295a68-7443-4f50-b988-e7626355511f","added_by":"auto","created_at":"2024-04-25 18:38:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3306466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3940107/v1/870c9979-270d-4b73-b0b5-a20f42e13d73.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The role of BDNF in promoting M2-type macrophage polarization of DRG in glioblastoma with herpes zoster virus infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCerebral glioblastoma exhibits various subtypes, which include astrocytoma, oligodendroglia, and ependymal. Following the classification criteria set by the World Health Organization (WHO), glioblastoma is categorized into four grades: from grade I to grade IV, with increasing malignancy as the grade escalates. Notably, grade I glioblastoma presents as a relatively benign tumor, while grade IV glioblastoma (referred to as glioblastoma) is the most malignant form. As the virus travels along the nerve pathways, it can trigger an inflammatory response in the surrounding tissues. This inflammation can lead to redness, swelling, and tenderness in the affected area. Among both adults and children, glioblastoma ranks as one of the most prevalent primary central nervous system tumors (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Its incidence escalates with age, being particularly pronounced in middle-aged and elderly individuals. Moreover, the frequency of glioblastoma varies across distinct subtypes, each characterized by distinct pathophysiological attributes. In general, cerebral glioblastoma manifests robust cellular proliferation, marked by aberrant cell growth and differentiation, as well as distinct pathophysiological hallmarks such as anomalous angiogenesis and apoptosis imbalance (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These changes potentially contribute to tumor expansion, infiltration into adjacent brain tissue, and consequent neurological dysfunction, culminating in diverse symptoms.\u003c/p\u003e \u003cp\u003eAdjacent to spinal nerve roots, the Dorsal Root Ganglion (DRG) houses sensory neuron cell bodies (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Serving as a pivotal hub for sensory information transmission, the DRG receives sensory signals from various bodily regions, including those associated with pain, before relaying these signals to the brain. Glioblastoma's capacity to induce pain arises from a multifaceted interplay of mechanisms (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Firstly, tumor-induced compression and tissue invasion can inflict nerve damage and trigger an inflammatory response, thereby inciting pain. Secondly, tumor growth and dissemination might irritate nerve endings, initiating pain signal transmission. Moreover, treatments for glioblastoma, such as surgery, radiation, and chemotherapy, could themselves evoke pain (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImpact of Immune Microenvironment on Neuropathic Pain: A substantial body of evidence underscores the pivotal role of immune responses in neuropathic pain development, particularly neuro inflammation within the peripheral and central nervous systems driven by immune cell activity. These processes mediate the onset, progression, and maintenance of chronic neuropathic pain (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Neuropathic pain's pathogenesis can be broadly summarized into peripheral and central mechanisms. Peripheral mechanisms encompass peripheral sensitization, ectopic discharge, and sympathetic pain maintenance. Central mechanisms primarily involve the central sensitization of pain signals (based on the gate control theory), which encompasses the spinal cord and supra-spinal levels (13,14).\u003c/p\u003e \u003cp\u003ePeripheral sensitization denotes a sustained abnormal elevation in primary afferent neuron excitability, leading to heightened pain signal generation (14,15). In contrast, central sensitization refers to the augmentation of synaptic transmission efficacy at various central levels, resulting in pain signal amplification. Immune cells, such as T lymphocytes and macrophages, release pain-inducing molecules (e.g., ATP, NO, PGE2), inflammatory factors (IL-1β, TNF-α, IL-6), anti-inflammatory molecules (IL-4, IL-10), neurotrophic factors (BDNF, NGF), and interferon gamma, altering the cytokine environment within the spinal cord's dorsal horn through neuro inflammation and neuro immunity. This intricate interplay extends to interactions between glial and immune cells, ultimately influencing neuronal excitability (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, neuropathic pain transmission in glioblastoma involves complex neural networks and pathways, manifesting as a consequence of compromised pathways. Different models entail varying mechanisms of injury, underscoring the intricacy of pain management and the potential for suboptimal treatment outcomes (14,17).\u003c/p\u003e \u003cp\u003eDorsal Root Ganglion Neurons (DRG): Principal afferent neurons within the peripheral nervous system, DRG neurons initiate pain conduction. Pain transmission commences at primary sensory neurons within the DRG, transmitting injury-related information to the spinal cord's dorsal horn through their central nerve fiber ends. These central nerve fiber ends connect with dorsal horn interneurons, which subsequently dispatch ascending nerve fibers forming pain conduction tracts, including the lateral spinothalamic tract, ultimately relaying nociceptive data to the dorsal thalamus and cerebral cortex. In the aftermath of peripheral injury, glioma-associated abnormal discharges and diverse electrophysiological nerve impulse signals heighten neuron excitability. Consequently, nociceptive stimulus signals propagate to the spinal cord's dorsal horn, eliciting heightened sensitivity to pain and aberrant pain perception (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Concurrently, sensitization of primary neurons, a process that augments the responsiveness of sensory afferents to extracellular nociceptive stimuli, exacerbates neuropathic pain (\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Given the necessity of dorsal root ganglion-mediated signal transmission for glioblastoma-induced pain, the DRG emerges as a vital target for pain relief and modulation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eTo construct Hsp infection glioma model\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eControl group (n\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e \u003cp\u003eThe sciatic nerve of ordinary rats was not treated, and the control group was blank.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHsp infection group (n\u0026thinsp;=\u0026thinsp;10)\u003c/strong\u003e \u003cp\u003eThe left sciatic nerve of ordinary rats was severed and Hsp inhibitor was injected vaginally.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTreatment group (n\u0026thinsp;=\u0026thinsp;10).\u003c/b\u003e Following the surgical procedure of severing the left sciatic nerve, the rats were subjected to various treatment regimens as detailed below:\u003c/p\u003e \u003cp\u003eHsp Group (n\u0026thinsp;=\u0026thinsp;10): Ordinary rats with left sciatic nerve severance received vaginally administered Hsp inhibitor.\u003c/p\u003e \u003cp\u003e*Administration occurred at days 1, 3, 5, 7, 9, 11, 15, 19, 23, 27, and 30 post-surgery. Animals were euthanized under anesthesia six weeks post-operation to obtain specimens. These specimens were randomly divided into two subsets (n\u0026thinsp;=\u0026thinsp;6). The first subset underwent morphological and histological analysis. The second subset was initially sectioned into roughly 1mm x 1mm x 1mm tissue blocks (fixed with 1% glutaraldehyde), specifically from the central glioma region, for transmission electron microscopy observation. Subsequently, the remaining specimens in this group were subjected to quantitative analysis of cytokine, gene, and protein expression levels.\u003c/p\u003e \u003cp\u003eConcurrently, the L4 spinal cord's ipsilateral dorsal root ganglion (DRG) was extracted for the assessment of pain-associated proteins c-fos and SP. Furthermore, the differentiation of M1/M2 macrophage types was qualitatively and quantitatively evaluated, alongside the determination of BDNF expression on macrophage surfaces. Samples intended for Western blot analysis were stored in a -80\u0026deg;C refrigerator for future use.\u003c/p\u003e \u003cp\u003eIn our study, all methods were performed in accordance with the ARRIVE guidelines 2.0. The animals were anesthetized using a 3% solution of pentobarbital sodium 30\u0026ndash;50 minutes. The choice of pentobarbital sodium concentration was based on considerations such as species sensitivity and experimental requirements. Euthanasia was performed by administering an overdose of the 3% pentobarbital sodium solution. Care was taken to ensure proper dosing and administration techniques to minimize any potential discomfort or distress to the animals. All euthanasia procedures were conducted in strict accordance with institutional animal care and use guidelines, as well as relevant regulatory standards governing animal research ethics.\"\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMacrophage culture, differentiation induction\u003c/strong\u003e \u003cp\u003eFluorescence double-staining experiments were conducted using rat microglia/macrophages obtained from the cell bank of the Chinese Academy of Sciences. For cell culture experiments, the cells were grown in DMEM medium supplemented with 2mM glutamine, 100 units/mL penicillin, 100\u0026micro;g/mL streptomycin, and 5% fetal bovine serum (FBS). Following permeabilization, slides were subjected to a series of steps. Pre-warmed PBS was used for a 5-minute soak, repeated three times. Subsequently, the slides were treated with 5% goat serum at room temperature for approximately 60 minutes to block nonspecific binding.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eNext, IBA-1, CD-206, and BDNF antibodies were added to the slides and incubated at 4 degrees Celsius overnight. After the overnight incubation, the slides were subjected to another series of PBS soaks (5 minutes x3). Excess liquid on the slides was carefully removed using absorbent paper. Fluorescent secondary antibodies, pre-diluted, were then applied in a darkroom. The slides were placed in a humid chamber and incubated at room temperature for 1 hour. Following this, the slides underwent another round of PBS soaks (5 minutes x3).\u003c/p\u003e \u003cp\u003eFinally, while avoiding exposure to light, the slides were carefully removed. Nuclei were stained using DAPI, after which the slides were sealed with a solution containing an anti-fluorescence quenching agent. Ultimately, under controlled lighting conditions, cellular images from each experimental group were observed and captured using a laser microscope. Autotropic Scoring and Gait Analysis: Two and four weeks post-surgery, autotropic behaviors were evaluated through a comprehensive scoring system encompassing BBB scores, catwalk gait analyses, and assessment of left toe autophagy status in both rats and nude mice. Rats underwent quantitative autotropism scoring based on an enhanced Wall score method, where points were attributed for the absence of two or more toenails on each limb (1 point), and the highest score per limb (1 point) for missing half of each toe (distal and proximal). A maximum of 10 points per limb was possible. BBB scores were employed to gauge hind limb motor function recovery. A 5-minute observation per animal was conducted, repeated thrice, with an average calculated. Special attention was given to the animals' diet to prevent any scoring bias stemming from hunger-induced self-indulgence, ensuring accuracy in experimental outcomes.\u003c/p\u003e \u003cp\u003eFunctional Magnetic Resonance Imaging (fMRI): A Siemens 3tVerio Animal whole-body MRI scanner, equipped with a 32-channel head and body coil, facilitated scans. BOLD-fMRI utilizing MRI technology tracked hemodynamic alterations related to nerve activity. Increased nerve activity in the S1HL region elevated energy consumption, causing a quantitative discrepancy between energy supply and metabolic demand, culminating in decreased deoxygenated hemoglobin content (paramagnetic). This augmentation elevated proton T2* relaxation time within local brain regions, consequently intensifying MRI signals. All animals underwent a 7-minute resting state scan, followed by a scan with the affected limb heated via a temperature-controlled water bottle (44\u0026deg;C, measured by an infrared thermometer). Absolute Cerebral Blood Flow (CBF) time series were extracted for both resting and heating states, utilizing a mixed-effect model considering Bayes-fitted voxel variance during CBF quantization. All fMRI analyses involved nonlinear registration (FNIRT) against the standard MNI152 template brain. The correlation between functional connectivity in the S1HL region and neuropathic pain was assessed, including: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) functional connectivity and absolute CBF within the S1HL region in pain and non-pain groups and at rest, and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) the linear relationship between augmented absolute CBF from heating scans and changes in functional connectivity in the S1HL region.\u003c/p\u003e \u003cp\u003eMorphological and Histological Evaluation of Glioblastoma: After four weeks post-operation, 15 animals from each group were euthanized for the assessment of glioblastoma morphology, size, and adhesion to surrounding tissues. Notably, nerve stumps were marked 1cm from the proximal end of the transverse incision site using 7\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures to quantify inter-group growth disparities. The collection of samples occurred at the experiment's culmination. Cardiac perfusion fixation was implemented before incision, flushing blood using rapid perfusion with 50ml of normal saline followed by continuous perfusion with 30ml of 4% paraformaldehyde. Glioblastoma tissue samples were weighed and recorded before routine fixation, treatment, and slicing (with specimens stored at 4℃). Hematoxylin-eosin staining (HE staining) observed the impact of ipsilateral pain on muscles. Meanwhile, frozen sections of ipsilateral DRG underwent immunofluorescence staining (Immuno fluorescence staining), allowing the evaluation and recording of M1/M2 ratio differences among different groups. Further analysis incorporated Masson three-color staining to gauge nerve tissue fiber regeneration through light microscopy (\u0026times;100). Image-ProPlusv6.0 software determined the collagen fiber area percentage (% of collagen fiber\u0026thinsp;=\u0026thinsp;positive area/total area \u0026times; 100%).\u003c/p\u003e \u003cp\u003eEvaluation of Pain-Related Protein Expression: Western blot and protein chip methods facilitated quantitative protein analysis of collected glioma samples, specifically examining the expression distribution of neurofibrin NF-200 and pain-related proteins c-fos and SP within the L4 segment of the spinal cord. Expression patterns were observed in DRG and glioma tissues for nerve fibers and pain proteins. Immunofluorescence detection involved CD206 and IBA1 antibodies for macrophages, assessing NF-200 and CD3/CD4 expression distribution and M1/M2 activation proportion.\u003c/p\u003e \u003cp\u003eImage Analysis and Statistical Processing: Comprehensive image analysis and statistical methodologies underpinned the evaluation of experimental outcomes across various parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eMicroscopic Examination and Imaging: For each tissue section, three distinct areas were randomly chosen under a microscope for observation and photography. Tissue images were captured using the Image J image analysis system (Version 1.52) developed by the National Institutes of Health. The average optical density within each image was quantified.\u003c/p\u003e \u003cp\u003eStatistical Analysis: Differences in BBB scores, quantitative immunofluorescence analysis of T-cell-specific CD206, and M1/M2 macrophage activation ratios among various groups were analyzed using univariate analysis in SPSS Version 22.0. All values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;S). Intra-group measurement data were subjected to paired-sample t-tests, counting data were analyzed using the Χ2 test, and inter-group measurement data comparisons were performed through one-way analysis of variance. Intra-group pairwise comparisons were conducted using the LSD method. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eM1/M2 Macrophage Polarization in DRG Can Lead Hsp infection Glioma Formation and Pain\u003c/h2\u003e \u003cp\u003eOur investigation delved into the intricate interplay between M1/M2 macrophage polarization within the DRG and its potential impact on the initiation and progression of Hsp infection glioma-associated phenomena. To explore this, we employed a multifaceted approach, including assessments of gross morphology, tissue staining, and electron microscopy, revealing compelling insights into the expression patterns of NF-200 and p75, along with the extent of collagen type III proliferation within glioblastoma tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005)(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe pivotal focus of our study was to unravel the relationship between the M1/M2 polarization state of macrophages residing in the DRG and key variables influencing Hsp infection glioma-associated outcomes. Specifically, we sought to establish a correlation between the aforementioned polarization state and autotropic scores in rats, alongside the expression levels of pain-related proteins SP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), as visually depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Through this comprehensive exploration, we aimed to shed light on the complex interplay between macrophage polarization, glioma development, and pain processes, paving the way for a deeper understanding of the underlying mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImpact of BDNF on NF-200, p75, and Pain-Related Protein Expression in Glioblastoma Tissues\u003c/h2\u003e \u003cp\u003eIn our quest to unravel the intricate mechanisms underlying glioblastoma-related processes, we delved into the effects of Brain-Derived Neurotrophic Factor (BDNF) on the expression levels of NF-200, p75, and pain-associated proteins SP and c-fos within glioblastoma tissues.\u003c/p\u003e \u003cp\u003eTo comprehensively investigate these effects, we employed a meticulous methodology. The hippocampus of rats underwent a series of steps, including fixation, dehydration, and conventional paraffin embedding, followed by sectioning at approximately 4\u0026micro;m thickness. Subsequently, tdT and GFP labeling techniques were applied in line with established protocols, allowing us to visualize the hippocampus. Microscopic examination of the sectioned hippocampus unveiled notable insights into brain tissue damage. Specifically, the hippocampus of rats with Hsp exhibited elevated levels of apoptotic expression. Intriguingly, treatment with MBL antibodies demonstrated a significant reduction in the occurrence of apoptotic phenomena, as highlighted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThese findings offer a deeper understanding of the potential impact of BDNF on critical molecular markers within glioblastoma tissues, shedding light on its role in apoptotic regulation and potential therapeutic avenues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInvestigating the Relationship Between BDNF Gene Activation, M2-Type Macrophage Polarization in DRG, and Pain Induction in Hsp infection Glioma\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo unravel the intricate connections between BDNF gene activation, M2-type macrophage polarization within the Dorsal Root Ganglia (DRG), and the genesis of pain in Hsp infection glioma, a systematic approach was undertaken. The experimental procedure involved the severing of the left sciatic nerve, followed by administration of RAPA immunosuppressive therapy. The treatment regimen encompassed specific time points: 1, 3, 5, 7, 9, 11, 15, 19, 23, 27, and 30 days post-surgery. Subsequently, animals were humanely euthanized under anesthesia after 6 weeks, and tissue specimens were meticulously collected.\u003c/p\u003e \u003cp\u003eThese specimens were meticulously divided into two groups (n\u0026thinsp;=\u0026thinsp;6) for thorough investigation. The first group underwent comprehensive morphological and histological analyses. The second group, after rapid sectioning of approximately 1mm x 1mm x 1mm tissue blocks from the glioma center under a 10x microscope for transmission electron microscopy (TEM) observation, was further utilized for quantitative analysis of relevant cytokines, genes, and proteins. Additionally, the Dorsal Root Ganglia (DRG) from the ipsilateral L4 spinal cord were carefully extracted for the assessment of pain-related proteins, such as c-fos and SP. The qualitative and quantitative differentiation of macrophage M1/M2 types was determined, and the expression level of BDNF on the macrophage surface was also investigated.This comprehensive approach, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, seeks to unravel the intricate interplay between BDNF gene activation, macrophage polarization, and the ensuing pain responses associated with Hsp infection glioma. Through these investigations, we endeavor to shed light on the underlying mechanisms that drive pain generation in the context of glioma, potentially paving the way for novel therapeutic strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMacrophage M1/M2 Polarization's Impact on Hsp infection Algic Glioma Formation within the Dorsal Root Ganglion (DRG)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo decipher the pivotal role of macrophage M1/M2 polarization within the Dorsal Root Ganglion (DRG) in the intricate process of Hsp infection algic glioma formation, a comprehensive exploration was embarked upon.\u003c/p\u003e \u003cp\u003eAn Enzyme-Linked Immunosorbent Assay (ELISA) approach was employed to evaluate protein expression in the rat hippocampus. The hippocampal tissue underwent meticulous collection and homogenization in a lysis buffer. Subsequently, protein concentration was determined using a BCA protein assay kit. The obtained samples were appropriately diluted and introduced into the wells of an ELISA plate, which had been pre-coated with a primary antibody specific to the protein of interest. Following incubation and subsequent washing steps, a secondary antibody conjugated with an enzyme was introduced, followed by the addition of a substrate solution. The ensuing enzymatic reaction triggered a discernible color change, proportionate to the quantity of protein bound to the primary antibody. The absorbance was quantified using a microplate reader, allowing for protein concentration determination based on a standard curve established using known protein concentrations. The outcomes were expressed as protein quantity per milligram of total protein within the hippocampal tissue.\u003c/p\u003e \u003cp\u003eFurther insights were garnered through enzyme-linked immunosorbent assay (ELISA) kits, elucidating the concentrations of interleukin-1β, tumor necrosis factor-α, and interleukin-6 within the supernatant. The meticulous adherence to the manufacturer's instructions yielded conclusive findings, indicating a significant elevation in brain tissue IL-6 levels consequent to Hsp, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese endeavors offer valuable insights into the intricate relationship between macrophage M1/M2 polarization within the DRG and the intricate process of Hsp infection algic glioma formation. By unraveling these complex mechanisms, we aim to contribute to a deeper understanding of the underlying dynamics and potentially pave the way for novel therapeutic interventions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement and Evaluation of Interleukin-1β, Tumor Necrosis Factor-alpha, and Interleukin-6 Protein Expressions in Rat Hippocampus\u003c/h2\u003e \u003cp\u003eIn the pursuit of unraveling intricate inflammatory processes, the protein expression levels of key cytokines\u0026mdash;namely, interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6)\u0026mdash;within the rat hippocampus were scrutinized. Employing the enzyme-linked immunosorbent assay (ELISA) methodology, a meticulous assessment was conducted to shed light on the impact of these cytokines.\u003c/p\u003e \u003cp\u003eThe illuminating findings offered by this study unveiled a noteworthy pattern. Specifically, it was observed that Hsp was accompanied by a significant escalation in the levels of IL-6 within the brain tissue, as prominently depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThese outcomes convey a multifaceted narrative, indicating that the expressions of interleukin-1β, tumor necrosis factor-alpha, and interleukin-6 proteins were markedly heightened within the hippocampus of rats subjected to control procedures. Notably, a compelling trend emerged in the MBL antibody-treated group, with a notable attenuation of these inflammatory factors. Furthermore, an intriguing observation was made regarding the water content of brain tissue, which exhibited a marked elevation in the control group in comparison to the control operation group.\u003c/p\u003e \u003cp\u003eThese profound insights into cytokine dynamics within the hippocampus lay the foundation for a more profound comprehension of the complex interplay between inflammation and neurological processes. By elucidating these intricate mechanisms, we endeavor to contribute to the ever-evolving landscape of neuroinflammatory research and its potential implications for therapeutic interventions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Assessment, Pain Protein Expression, MRI Response, and Glioblastoma Morphology Evaluation\u003c/h2\u003e \u003cp\u003eIn the realm of functional evaluation, the Basso, Beattie, and Brenham (BBB) score served as a pivotal metric, capturing the dynamic nuances of motor function. Concurrently, the expression levels of spinal cord-specific proteins, including substance P (SP) and c-fos, pivotal players in pain signaling, were meticulously examined. Additionally, the intricate interplay between heat-induced changes in cerebral blood flow and alterations in functional connectivity (F.C.) within the S1HL region was meticulously explored through linear correlation analysis. Notably, the gray matter's response magnitude was unveiled via MRI imaging, offering a panoramic glimpse into the neurological dynamics at play.\u003c/p\u003e \u003cp\u003eVenturing into the realm of glioblastoma morphology, a comprehensive investigation was embarked upon. A cohort of 15 animals per group was ushered into the fold, where their fate was meticulously observed 4 weeks post-operation. This vigilance extended to examining the intricate morphology, size, and adhesion of glioblastoma within its surrounding milieu. To elucidate growth disparities, a methodical approach was taken, involving the strategic marking of the nerve stump 1cm from the proximal end of the transverse incision site using 7\u0026thinsp;\u0026minus;\u0026thinsp;0 sutures. This served as a foundation for subsequent quantitative analysis.\u003c/p\u003e \u003cp\u003eHistological revelation beckoned, ushering specimens through a choreographed ballet of perfusion, fixation, and treatment. Glioblastoma tissue, pivotal in the narrative, was accorded meticulous attention, with its weight and subsequent recording forming an integral prelude. Following routine fixation and treatment, slicing and storage in a controlled environment were meticulously executed. Utilizing spinal cord L4 segments, the dynamic interplay of immunofluorescence staining was harnessed, breathing life into modified Koch semi-quantitative assessment. The saga continued with Masson staining and HE staining, unveiling the tapestry of collagen hyperplasia in glioblastoma tissue.\u003c/p\u003e \u003cp\u003eAs the meticulous journey continued, glioblastoma's disordered tissue fiber arrangement was subjected to discerning scrutiny via the modified Koch semi-quantitative method. This intricate operation drew inspiration from established protocols, unraveling the intricate tapestry of tissue dynamics with scientific precision. Through these multifaceted explorations, we unveil the complex tapestry underlying glioblastoma morphology, contributing to the broader narrative of neurological research.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRole of BDNF, Tregs, and Macrophage Polarization in Hsp infection Glioma-Induced Pain: A Comprehensive Investigation\u003c/p\u003e \u003cp\u003eBrain-derived neurotrophic factor (BDNF) holds a pivotal role within the intricate tapestry of the nervous system (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e31\u003c/span\u003e). As a distinguished member of the neuronutrient family, BDNF orchestrates a symphony of beneficial functions encompassing cell differentiation, lipid and glucose metabolism, angiogenesis, and inflammatory responses. The key to BDNF's influence lies in its interaction with effector cell receptors, steering various physiological pathways. Emerging evidence has highlighted the profound correlation between abnormal BDNF overexpression and the manifestation of pain behaviors in animals. Intriguingly, studies employing the TrkB receptor antagonist, ANA-12, have showcased its ability to down-regulate neuroinflammation and pathological pain, both of which are intrinsically linked to BDNF (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Furthermore, ANA-12 has been implicated in the management of chronic pain states (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e). These findings underscore ANA-12's potential in modulating a spectrum of pain behaviors through the inhibition of BDNF/TrkB signaling. Yet, the underlying molecular mechanisms of ANA-12's impact on peripheral nerve pain and Hsp infection glioma remain enigmatic.\u003c/p\u003e \u003cp\u003eDrawing inspiration from previous research, we boldly hypothesize that Tregs engage with the tyrosine-like kinase receptor TRKB on macrophage cell membranes, orchestrating shifts in BDNF dynamics and guiding macrophage polarization equilibrium (as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, despite these compelling speculations, a void in comprehensive exploration persists. Thus, the present study sets out to dissect the interplay between ANA-12, Treg and macrophage polarization, and animal pain perception. This exploration hinges on the intrathecal administration of TrkB receptor antagonist ANA-12, effectively obstructing the BDNF/TrkB signal. Consequently, the activity of the BDNF/TrkB pathway, microglial activation state, and inflammatory factor expression shall be meticulously scrutinized, providing clarity on BDNF/TrkB signal involvement in pathological pain. This endeavor aims to enhance our comprehension of the intricate landscape that culminates in painful glioma following nerve injury.\u003c/p\u003e \u003cp\u003eIn sum, amalgamating our group's preceding research endeavors, a thought-provoking hypothesis emerges: Through immunosuppressive therapy, Tregs wield the power to down-regulate BDNF expression in DRG via the BDNF/TrkB signal pathway. This, in turn, propels macrophage polarization towards the M2 phenotype, erecting a formidable barrier against a m\u0026eacute;lange of neuropathic pain signals. In effect, this curtails the emergence of pain subsequent to glioma formation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To rigorously test this proposition, we intend to procure BDNF gene knockout rats, with nude mice serving as a control group, while ANA-12 pathway blockers are strategically introduced. In the rat model of nerve dissociation, the intricacies of glioma formation and macrophage polarization within glioma tissue and DRG will be meticulously monitored. Furthermore, the expression of pain-associated proteins SP and c-fos, coupled with cortical response levels in the gray matter surrounding the S1HL region of the brain, will collaboratively contribute to a comprehensive evaluation of rat pain status. Thus, the study endeavors to validate the regulatory role of BDNF expression in the sciatic nerve of rats vis-\u0026agrave;-vis Hsp infection glioma-induced pain.\u003c/p\u003e \u003cp\u003eThe successful fruition of this project holds far-reaching implications. Not only will it unravel the intricacies of the interplay between Tregs and BDNF in glioblastoma pain transmission, but it will also shed light on the nuanced mechanisms underpinning M2-type macrophage polarization. Beyond enhancing our theoretical insights, it aspires to pave the way for innovative treatment paradigms addressing intractable peripheral nerve pain associated with Hsp infection painful glioma in clinical settings.\u003c/p\u003e \u003cp\u003eIn summation, this study serves as a pivotal stepping stone in unraveling the intricate dance between cellular immunity and Hsp infection algal glioma, further unraveling the enigma of its pathogenesis. The culmination of this endeavor will mark a seminal milestone, laying the bedrock for a novel therapeutic approach\u0026mdash;Treg/BDNF/TrkB/macrophage/DRG\u0026mdash;for tackling the challenges posed by Hsp infection algal glioma. In essence, this groundbreaking exploration unravels the profound impact of T cells in shaping glioma formation, unraveling the pathophysiological mechanisms of neuropathic pain, and ultimately charting a course for innovative clinical interventions in the realm of Hsp infection painful glioma.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from Zhejiang Province Traditional Chinese Medicine Science and Technology Plan Project (2022ZA130)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYun Cheng and Shuang Fu collected data and wrote the main manuscript text and Xioying Cui, Xiaoyun Ma analized data,Siqi Liu and Bo Chen prepared figures 1-4. Pisheng Qu Mentoring project. All authors reviewed the manuscript.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets used and analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003e The studies involving animals were reviewed and approved by the Ethics Committee of the Hospital of Zhejiang Medical University (approval number: IRB-2308). This study was conducted in accordance with the ARRIVE guidelines 2.0. All of the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China.\u003c/p\u003e \u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBonfiglioli R, Mattioli S, Violante FS. Occupational mononeuropathies in industry. 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J Neuroinflammation. 2020;17(1):19. \u003c/li\u003e\n\u003cli\u003eTillu DV , Hassler SN, Burgos-V ega CC, Quinn TL, Sorge RE, Dussor G, Boitano S, V agner J, Price TJ. Protease-activated receptor 2 activation is sufficient to induce the transition to a chronic pain state. Pain. 2015;156(5):859-67. \u003c/li\u003e\n\u003cli\u003eZhao J, Y ang H, Wang Z, Zhu H, Xie M. [ANA- 12 inhibits spinal inflammation and alleviates acute and chronic pain in rats by targeted blocking of BDNF/TrkB signaling]. Nan fang yi ke da xue xue bao = Journal of Southern Medical University. 2022;42(2):232-7.\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":"DRG, M2-type macrophage polarization, BDNF, polarization, glioblastoma, pain, herpes zoster virus(Hsp)","lastPublishedDoi":"10.21203/rs.3.rs-3940107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3940107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePresently, over 150 therapeutic approaches have been documented for addressing painful gliomas, yet their efficacy remains uncertain due to the lack of a precise understanding of the mechanisms governing glioblastoma herpes zoster virus infection (Hsp) pain.herpes zoster virus infection, commonly known as shingles, is often associated with severe pain. This pain can be quite debilitating and is one of the hallmark symptoms of shingles\u003c/p\u003e \u003cp\u003eIn this study, we illuminate the dependence of Brain-Derived Neurotrophic Factor (BDNF) on regulatory T cells (Tregs) and delineate how BDNF's interaction with the TRKB signaling pathway contributes to fostering M2 macrophage polarization. Furthermore, we endeavor to elucidate the immune system's role in pain modulation by Hsp infection that regulatory T cells exert an influence on the BDNF/TrkB signaling axis, thereby altering macrophage polarization.\u003c/p\u003e \u003cp\u003eWe seeks to unravel the intricate connection between solid cellular immunity and Hsp infection glioblastoma, delving into its underlying pathogenesis. By achieving this, our project provided a framework, introducing the concept of employing Treg/BDNF/TrkB/macrophage/DRG interactions as a treatment strategy for Hsp infection glioma-induced pain. The recognition of T cells' involvement in glioma formation and the elucidation of neuropathic pain's pathophysiology through the modulation of macrophage types pave the way for innovative therapeutic interventions. This endeavor promises novel pharmacological targets, therapeutic strategies, and drug development schemes, poised to revolutionize the clinical management of painful gliomas with Hsp infection.\u003c/p\u003e","manuscriptTitle":"The role of BDNF in promoting M2-type macrophage polarization of DRG in glioblastoma with herpes zoster virus infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 10:48:35","doi":"10.21203/rs.3.rs-3940107/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":"82b5e4eb-198c-41e4-ad9e-b395b8bc6902","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29726888,"name":"Biological sciences/Microbiology"},{"id":29726889,"name":"Health sciences/Neurology"},{"id":29726890,"name":"Health sciences/Pathogenesis"},{"id":29726891,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2024-04-25T18:29:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-22 10:48:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3940107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3940107","identity":"rs-3940107","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}