A dopamine D1-like receptor agonist ameliorates stab wound-induced brain injury through its immunosuppressive effect

A dopamine D1-like receptor agonist ameliorates stab wound-induced brain injury through its immunosuppressive effect | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A dopamine D1-like receptor agonist ameliorates stab wound-induced brain injury through its immunosuppressive effect Mohammed E. Choudhury This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6726043/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 Traumatic brain injury (TBI) causes progressive nervous tissue degeneration long after the initial injury due to secondary neuroinflammatory reactions. G protein-coupled dopamine D1-like receptors, which elevate intracellular cAMP levels, have been shown to mediate the suppressive effects on lipopolysaccharide (LPS)-induced proinflammatory activation of microglia and macrophages. The present study investigated whether or not the D1-like receptor-specific agonist SKF-81297 (SKF) administered intraperitoneally once daily for 7 days starting 1 h after TBI could ameliorate TBI in a rat model of stab wounds in the forebrain. SKF reduced the volume of TBI-induced brain tissue loss, increased mobile activity, and ameliorated cognitive dysfunction two months after TBI. A single dose of SKF suppressed the expression of IL-1β and TNFα in brain tissue by reducing oxidative injury 24 h post-TBI. SKF decreased the expression of NADPH oxidase 2 subunits but did not affect antioxidative enzymes. SKF also prevented LPS-induced translocation of NFκB into the nuclei of macrophages. The agonist clenbuterol (CLB) for adrenergic β2 receptor, another Gs-linked GPCR, exerted comparable ameliorative effects in TBI model rats by suppressing neuroinflammation. In summary, SKF may exert anti-inflammatory effects by suppressing the NFκB pathway, similar to CLB, leading to amelioration of TBI-induced brain degeneration. Traumatic brain injury microglia macrophage SKF-81297 LPS NFκB ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights Microglia and macrophages in stab wound-induced traumatic brain injury (SW-TBI) expressed dopamine receptors. A dopamine D1-like receptor agonist ameliorated SW-TBI-induced behavioral deficits. The agonist inhibited cellular ROS generation and NFκB nuclear translocation. Gs-linked GPCRs mediate suppressive effects on TBI-induced neuroinflammation. Introduction Traumatic brain injury (TBI) is a leading cause of severe brain dysfunction, including motor, sensory, mental, and cognitive deficits, as well as acute mortality 1 , 2 , 3 , 4 . Even more worrying is the increasing number of stab wound-induced traumatic brain injury (SW-TBI) caused by knives and firearms, which directly damage blood vessels and brain tissue, causing intracerebral hemorrhages 5 , 6 . Numerous studies have demonstrated that chronic brain dysfunction resulting from TBI is closely associated with neuroinflammation, secondary to TBI-induced primary neural cell death. In this secondary neuroinflammation, resident microglia and infiltrating leukocytes (monocytes, granulocytes, and lymphocytes) play crucial roles in exacerbating pathophysiological processes in and around the injured tissues 1 , 2 , 7 , 8 . These immune cells release various proinflammatory mediators, including cytokines, reactive oxygen species (ROS), and nitric oxide. Monocyte-derived macrophages and activated microglia also accelerate neural cell death through phagocytic activity that is a process known as phagoptosis 9 , 10 . In addition to their detrimental roles, microglia and circulating monocyte-derived macrophages may contribute to preventing degeneration and promoting the regeneration of injured tissues by eliminating degenerated materials and releasing anti-inflammatory cytokines and neuroprotective growth factors 8 , 11 , 12 , 13 . Furthermore, these cells have the potential to scavenge ROS while suppressing ROS-induced cell death, including ferroptosis 14 , 15 , 16 . Single-cell RNA-sequencing data from a recent report using stab wound brain injury showed striking and distinct transcriptional changes in glial cell populations 17 . Therefore, interventions that suppress the detrimental nature and promote the beneficial functions of these immune cells accumulating in the injured tissues are of great interest. To date, various pharmacological interventions for TBI have been explored to suppress the detrimental effects and promote the beneficial functions of immune cells, particularly microglia and macrophages 7 , 18 , 19 , 20 , 21 . Methylprednisolone, a corticosteroid studied for its potential to reduce inflammation TBI, has been administered to patients with SW-TBI 20 , 21 . N-acetylcysteine, an antioxidant that may help reduce oxidative stress, has been investigated for its ameliorative effects in both laboratory 22 , 23 and clinical settings 24 , 25 . In our previous study, classical hypnotic bromvalerylurea (BU), which has anti-inflammatory effects on microglia and macrophages in addition to enhancing GABAA receptor-mediated neurotransmission, markedly improved outcomes in rats with SW-TBI 7 , 19 , 26 . BU exerts anti-inflammatory effects by activating Nrf2 [12] and by inhibiting JAK1 27 . Agents that increase intracellular cAMP levels have been reported to suppress the proinflammatory activation of microglia 28 , 29 , 30 . In particular, β2 adrenergic receptor (AR) agonists have strong immunosuppressive effects on lipopolysaccharide (LPS)-induced proinflammatory activation of microglia 28 , 31 . Microglia also express another G protein-coupled receptor (GPCR), the dopamine D1-like receptor, which is linked to Gs proteins 30 , 32 , 33 . Dopamine and the D1-like receptor-specific agonist SKF-81297 (SKF) suppress LPS-induced IL-1b and TNFa expression while increasing cAMP levels in primary rat microglia 30 , 32 . Given the strong immunosuppressive effects of SKF, we investigated whether or not SKF ameliorates SW-TBI in a rat model by suppressing the proinflammatory activities of microglia and infiltrating leukocytes. Results Expression of dopamine receptors Dopamine receptors D1 (DR1) and DR5 are classified as D1-like receptors, which are GPCR linked to G protein Gs. mRNA encoding DR1 and DR5 was expressed in the striatum of control, Vcl-, and SKF-81297 (SKF)-injected SW-TBI rats (Fig. 1 A). The D1-like receptor-specific agonist SKF suppressed LPS-induced IL-1b and TNFa expression in primary rat microglia and peritoneal macrophages (Supplementary Fig. 1). The injured core and its surrounding tissue were enzymatically dispersed in single cells, which were subjected to an FCM analysis using antibodies against CD11b and CD45 (Fig. 1 B). Infiltrated macrophages were identified as CD11b + /CD45 high cells and resident microglia as CD11b + /CD45 low cells. Both cell lines were sorted to prepare the total RNA for qPCR. The expression of mRNA encoding DR1, DR3, and DR5 was detected in both sorted microglia and macrophages (Fig. 1 C). Ameliorative effects of SKF on SW-TBI SKF was administered to SW-TBI rats once daily for 7 days, and its ameliorative effects were evaluated (Fig. 2 ). Probably due to secondary degeneration 1 , 2 , SW-TBI generates severe brain tissue loss on a monthly basis 7 . SKF suppressed brain tissue loss (Fig. 2 A). The total distance moved in the open-field test (OFT) decreased in SW-TBI rats, but SKF reversed this reduction (Fig. 2 B). SW-TBI has been shown to disturb cognitive and learning/memory functions 7 . The MWM test showed that SW-TBI did not cause apparent changes in the total distance moved, but SKF increased the frequency of entering the target quadrant in the pool, where the platform was placed under the waterline on the previous day (Fig. 2 C). This suggests that SW-TBI-induced cognitive and learning abilities were ameliorated by SKF. Effects of SKF on proinflammatory responses SW-TBI increased expression of IL-1b and TNFa at mRNA and protein levels in and around the injured core (Figs. 3 A and 3 B, respectively). SKF suppressed SW-TBI-induced elevated expression of these cytokines. FCM analyses showed that the percentage of total CD11b + cells decreased in injured brain tissues, while macrophages and granulocytes increased (Fig. 3 C). SKF reversed SW-TBI-induced changes in the CD11b + cell population. The microglia and macrophages were further characterized using FCM. SKF decreased CD11b expression in both microglia and macrophages (Fig. 3 D), whereas CD45 expression decreased in microglia but not in macrophages (Fig. 3 E). Forward scatter (FS) values also decreased only in microglia (Fig. 3 F). Microglia and macrophages were sorted from injured tissues at 1 dpi, and total RNA was isolated for qPCR. SKF administration decreased the mRNA expression of proinflammatory mediators IL-1b, TNFa, and CD86 in microglia (Fig. 3 F) but not in macrophages (Fig. 3 G). Suppression of NFkB-mediated proinflammatory pathways by SKF ROS cause the generation of 8-OHdG, a marker of oxidative damage to DNA. To determine whether or not SKF reduced oxidative stress in SW-TBI tissues, 8-OHdG levels in injured tissues dissected at 1 dpi were measured by an ELISA. SW-TBI significantly increased 8-OHdG levels, which were reduced by SKF (Fig. 4 A). DHE is oxidized to emit red fluorescence from ethidium 7 . CD11b + cells were sorted using MACS and analyzed by flow cytometry to measure oxidized DHE-derived fluorescence (Fig. 4 B). The mean fluorescence intensity was reduced in CD11b + cells sorted from SKF-treated rat brains compared to vehicle (Vcl)-treated rat brains. However, SKF-induced reduction of DHE-derived fluorescence by CD11b + cells was not marked, suggesting that CD11b − cells may be responsible for ROS generation. An immunohistochemical analysis showed that most Iba1 + /CD45 − ramified microglial cells did not exhibit red fluorescence, and Iba1 + /CD45 + round macrophages were weakly positive for DHE-derived fluorescence (Fig. 4Ca, Supplementary Fig. 2A). Furthermore, the study suggests that Iba1 − /CD45 − cells are the major source of ROS. Compared to Vcl-treated brain sections, SKF-treated sections displayed much weaker red fluorescence (Fig. 4Cb), suggesting that SKF might have suppressed ROS generation by not only myeloid cells but also neuroectodermal cells, such as neurons 7 . To reveal the mechanisms by which SKF reduced oxidative injury, the expression of oxidative and anti-oxidative enzymes was investigated by qPCR (Figs. 4 D and 4 E). The expression of gp91phox, a subunit of NADPH oxidase 2 (NOX2), was upregulated in Vcl-treated injured tissues, and this upregulation was reversed by SKF administration (Fig. 4 D). The expression of p47phox and p22phox, subunits necessary for NOX2 activity, was also increased by SW-TBI and suppressed by SKF. Conversely, the expression of antioxidative enzymes glutathione peroxidase 4 (GPX4), Cu/Zn superoxide dismutase (Cu/ZnSOD), and catalase was not elevated in injured tissues, and SKF did not affect (GPX4) or decrease the expression of Cu/ZnSOD and catalase (Fig. 4 E). ROS have been shown to induce NFκB activation, a critical transcription factor that elevates the expression of various proinflammatory factors, such as IL-1b and TNFa 34 , 35 . Immunoblotting revealed that LPS induced NFκB p65 localization in the nuclear fraction of rat peritoneal macrophages (Fig. 4 F). SKF prevented LPS-induced nuclear localization of p65. Phosphorylated IkB kinase (pIKK) induces the degradation of IkB, resulting in nuclear localization of p65 31 . To examine whether or not SKF suppressed the SW-TBI-induced nuclear localization of p65 in injured brain tissues, immunoblotting was performed to detect pIKK in injured tissues. SW-TBI caused phosphorylation of IKK in injured tissues, and SKF administration prevented this phosphorylation (Fig. 4 G). To determine the cell type bearing pIKK in and around injured tissues, an immunohistochemical study was conducted. Figure 4Ha shows that the pIKK + /CD11b + cells with morphological characteristics of infiltrated macrophages that are a round morphology without apparent ramified processes 13 . In contrast, ramified CD11b + cells or microglia appeared to be pIKK − . In SKF-treated rat tissues, the number of pIKK + cells was much lower than that in Vcl-treated rat tissues (Fig. 4Hb). In addition to microglia/macrophages, an abundant number of CD11b − cells appeared to be pIKK + (Supplementary Fig. 2b). Pro-inflammatory responses in immune cells are correlated with metabolic changes. SKF was found to suppress both anaerobic glycolytic and aerobic mitochondrial metabolism, as revealed by a flux analysis (Supplementary Fig. 3). Effects of SKF on anti-inflammatory, phagocytic, and neuroprotective responses The phagocytic activity of microglia and macrophages may be responsible for tissue restoration, as injury-induced accumulation of degenerated materials prevents restoration 11 . Conversely, it has also been proposed that viable cells are killed by the phagocytosis of microglia and macrophages. This aggravating process is called phagocytosis and is mediated by the opsonization of damaged cells with complement C1q 36 . SW-TBI increased the expression of CD68, a marker for phagosomes, and complement C1qb, and SKF reduced the increased CD68 and C1qb expression in the injured brain tissues (Fig. 5 A). SW-TBI increased the expression of the typical anti-inflammatory cytokine TGFb1 13, 37 , but SKF did not affect TGFb1 expression in the brain (Fig. 5 ). IGF1 is a neuroprotective factor involved in the restoration of injured brains, and blood-borne infiltrating macrophages may be a source of IGF1 12, 38, 39 . SW-TBI did not affect IGF1 expression, but SKF tended to reduce IGF1 expression. An in vitro study showed that SKF did not affect CD68 expression in cultured rat primary microglia or peritoneal macrophages (Fig. 5 B and 5 C). However, SKF reduced C1qb expression in microglia cultured in the presence of LPS. SKF reduced microglial TGFb1 and IGF1 expression in both the absence and presence of LPS. Similar suppressive effects of SKF on TGFb1 and IGF1 expression by macrophages were only observed in the absence of LPS because LPS strongly suppressed TGFb1 and IGF1 expression. These data suggest that the ameliorative effects of SKF are independent of the immunosuppressive cytokine TGFb1 and the neuroprotective factor IGF1. Effects of SKF on comprehensive gene expression in the injured brain tissues. RNAseq was conducted to comprehensively investigate gene expression, and the results are presented in a Venn diagram, illustrating the expression changes of 7,343 genes induced by SW-TBI. The expression of 72 genes was altered by SKF treatment. The primary genes with altered expression were a group of proinflammatory mediators, such as IL-1b (Fig. 6 A). A pathway analysis was performed to identify the specific pathways through which SKF exerted its effects. The analysis revealed that SKF inhibited pathways that promote immune cell activation and leukocyte infiltration into the brain (Fig. 6 B). A volcano plot demonstrated that the expression of many proinflammatory factors, such as IL-1b and NOS2, was decreased by SKF treatment (Fig. 6 C). Effects of the b2AR-specific agonist clenbuterol (CLB) on SW-TBI The effects of CLB, a BBB-permeable b2AR-specific agonist 40 , on SW-TBI were investigated. b2AR is a Gs-coupled GPCR identical to dopamine D1-like receptors 41 . CLP increased motor activity of SW-TBI rats (Fig. 7 A). CLB had an inhibitory effect on macrophage and granulocyte accumulation (Fig. 7 B). CLB also suppressed CD11b and CD45 expression in microglia in and around injured tissues (Fig. 7 C and 7 D). A qPCR analysis of gene expression around injured tissues revealed that CLB suppressed the mRNA expression of IL-1b, TNFa, and p22phox but did not affect Cu/ZnSOD expression (Fig. 7 E). Discussion SW-TBI is still an intractable condition, for which there are still no specific and effective interventions. SW-TBI has a high mortality rate, and most survivors suffer from neurological and psychological sequelae, which often severely disturb their well-being in daily life. Although microglia themselves may not be thought to have strong deleterious effects on the brain 7 , 8 , they can induce infiltration into the injured tissues of circulating leukocytes, especially monocytes, which are the precursor cells of brain macrophages 1 , 2 , 42 . The infiltrated leukocytes, in particular monocyte-derived macrophages, play aggravating effects on injured brains through several mechanisms: releasing pro-inflammatory mediators, such as proinflammatory cytokines/chemokines; generating ROS causing oxidative injuries in brain tissues and cells; and eliminating viable cells by phagocytosis 7 , 8 , 9 , 10 , 11 , 38 . Thus, microglia are thought to play a central role in the pathogenesis of SW-TBI. Although both microglia and macrophages express D1R and D5R, SKF may act more strongly on microglia than macrophages, and it more markedly suppresses the pro-inflammatory activation of microglia than macrophages 32 . SKF reduced the infiltration of monocytes and granulocytes, probably due to the reduced expression of proinflammatory mediators by microglia, resulting in the improvement of SW-TBI pathogenesis. SKF ameliorated the oxidative damage in and around the injured tissue caused by SW-TBI by suppressing the metabolic activity of microglia and macrophages and ameliorating the chronically progressive loss of brain tissue and cognitive dysfunction caused by SW-TBI, as well as motor inactivity. These results strongly suggest that pharmacological intervention to activate D1-like receptor-mediated signaling in SW-TBI pathology may improve the prognosis. RNAseq results showed that SKF suppressed the expression of proinflammatory mediators, such as IL-1b, which was driven by NFκB 13 , 31 . SKF may suppress translocation of the proinflammatory transcription factor NFκB into the nuclei to prevent proinflammatory responses in the injured brain. SKF reduced oxidative stress-induced injury, which was likely correlated with decreased proinflammatory reactions. SKF suppresses NOX2 enzyme subunit expression The present immunohistochemical observations suggest that CD11b − neural cells may be a critical source of ROS. Neurons express NOX2 during inflammation, chronically causing neurodegeneration 43 . SKF may have suppressed neuronal ROS generation. In contrast, SKF did not increase the expression of the anti-oxidative enzymes GPX4 or Cu/ZnSOD. These results suggest that although SKF suppresses NFκB, it is unlikely to increase the activity of the transcription factor NF-E2-related factor 2 (Nrf2), which increases the expression of antioxidant factors and enhances anti-inflammatory effects 18 . Regarding the mechanism of suppression of NFκB activity by SKF, it is well known that when intracellular cAMP concentration is elevated by b2AR agonists 28 or phosphodiesterase inhibitors 29 , 30 , nuclear translocation of NFκB is prevented [24]. As both D1-like receptors and b2AR are Gs-coupled GPCRs 33 , it is likely that they also inhibit the nuclear translocation of NFκB via an increase in intracellular cAMP concentration. In fact, this study showed that the b2AR agonist CLB exerted anti-inflammatory effects similar to SKF, suggesting that the ameliorating effects of SKF on SW-TBI were mediated by its cAMP-elevating effects on microglia and macrophages 44 . Microglia and infiltrating macrophages are not always detrimental to injured brains 12 , 13 , 38 . These cells release several neuroprotective cytokines and growth factors, such as IGF1, BDNF, and bFGF. They also secrete anti-inflammatory cytokines, including TGFβ1, which have strong and sustained effects in suppressing the proinflammatory activation of infiltrating macrophages. Thus, microglia and macrophages are often described as a "double-edged sword" 44 . Given the above, the ameliorative effects of SKF on SW-TBI may be attributed to the enhancement of the neuroprotective and anti-inflammatory actions of microglia and macrophages. However, as shown in this study, SKF decreased TGFβ1 and IGF1 expression. Stimulative effects of SKF on the expression of immunosuppressive or neuroprotective factors were not observed in the RNAseq study. Thus, it is likely that SKF ameliorates the prognosis of SW-TBI by suppressing SW-TBI-induced neuroinflammation through the inhibition of nuclear localization of NFκB. A concise visual representation of the primary findings of the study is presented in (Fig. 8 ). In conclusion, SKF, a blood-brain barrier-permeable dopamine D1-like receptor agonist, suppresses brain inflammation and improves the functional prognosis in a rat SW-TBI model. These results suggest that D1-like agonists are effective for the treatment of brain injury, a refractory pathology. This action was thought to be associated with an elevation of the intracellular cAMP concentration and appeared through the inhibition of the expression of proinflammatory mediators. Materials and Methods Animals All rats received standard care, and our study protocol was approved by Ethics Committee for Animal Experimentation of Ehime University, Japan (approval number #05U50-2). In accordance with the previous reports of our university, all approaches accomplished in this study strictly followed to the ARRIVE guidelines. Our animal facility-bred 10- to 12-week-old male Wistar rats weighing from 250 to 300 g (Clea Japan, Tokyo, Japan) were used in this study. The SW-TBI model was prepared by inducing stab wounds in the rat brain as described elsewhere 7 . In brief, under deep anesthesia with isoflurane (Mylan Pharmaceutical company, Tokyo, Japan), the rat's head was secured in a stereotaxic apparatus (Narishige, Tokyo, Japan), and a longitudinal incision of approximately 15 mm was made in the skin to expose the skull. Two holes were drilled through the skull over the right hemisphere at approximately 2.5 and 4 mm right of the midline and 1 mm posterior to the bregma, respectively. A 26-gauge needle was inserted through each hole to a depth of approximately 7 mm from the surface of the skull and moved in a fan-like manner from anterior to posterior, parallel to the midline. The needle was then withdrawn, and the skin incision was closed with quick-drying glue (Aron-Alpha; Toagosei, Tokyo, Japan). Soldem 3A solution (10 ml/rat/day; Terumo Corporation, Tokyo, Japan) was administered for postoperative care. For Euthanasia, CO2 (Matsuyama Nishi Sanso Company, Matsuyama, Japan) inhalation exposure was performed where CO₂ flow rate was maintained to replace 30–50% of the cage volume per minute 45 , 46 . Primary microglia and macrophage cell cultures Rat primary microglial cell cultures were prepared as previously described 47 , where cortices from newborn rat pups were mechanically dissociated into individual cells. Dissociated cells were cultured as mixed glial cultures in 75 cm 2 flasks with 10% fetal calf serum (FCS)-supplemented Dulbecco’s Modified Eagle’s medium (DMEM; Fujifilm Wako, Osaka, Japan). After 11 days, microglial cells were obtained by agitating the flasks at 200 rpm for 1 h at 37°C, and pure microglial cells were plated in poly L-lysine-coated 6-well culture plates for an additional 11 days. Rat primary macrophages were harvested from the peritoneal lavage of adult rats as described elsewhere 48 and cultured in DMEM supplemented with 3% FCS-supplemented DMEM (Fujifilm Wako). For pharmacological studies, the culture medium was removed, and the cells were incubated for 6 hours in serum-free E2 medium (DMEM containing 10 mM HEPES, pH 7.3; Gibco, Grand Island, NY, USA; 4.5 mg/mL glucose, Gibco; 5 µg/mL insulin, 5 nM sodium selenite, 5 µg/mL transferrin, Gibco; and 0.2 mg/mL bovine serum albumin, Sigma-Aldrich, St. Louis, MO, USA). Inflammatory cell culture models were prepared by exposing the cells to E2 medium containing 1 µg/mL LPS (from Escherichia coli , serotype 055:B5; Sigma-Aldrich) for 6 h or overnight, as required by each assay. Pharmacological interventions In Vivo Studies : The D1-like receptor-selective agonist SKF-81297 hydrobromide (SKF; Tocris Bioscience, Bristol, UK) was dissolved in DMSO and diluted with normal saline to a concentration of 2.5%. Normal saline containing 2.5% DMSO was used as the Vcl. SKF was administered intraperitoneally to SW-TBI rats at a dose of 10 mg/kg, 1 h after SW-TBI. In the long-term experiments, SKF was administered once per day for 7 days at the same dose. Clenbuterol hydrochloride (CLB; Sigma-Aldrich), dissolved in the same manner as SKF, was administered subcutaneously at a dose of 0.1 mg/kg body weight, following the same administration schedule as SKF. In Vitro Studies : LPS-treated and untreated cells were exposed to 10 µM SKF. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) Brain tissue from the lesion area (approximately 100 mg) of SW-TBI and normal rats was dissociated into single cells using a gentleMACS dissociator with 37C_ABDK (Miltenyi Biotec, Tokyo, Japan) and an adult brain dissociation kit (Miltenyi Biotec), as previously described 7 . The prepared cell suspensions were subjected to FCM analyses and FACS. For multicolor immunofluorescence labeling, the cells were blocked with mouse anti-rat CD32 antibody (Rat BD Fc Block; BD Biosciences, Franklin Lakes, NJ, USA), followed by labeling with fluorescent antibodies against cell surface antigens by incubating the cells for 30 min at 4°C using the antibodies listed in Supplementary Table 1. Cells labeled with antibodies for flow cytometry were analyzed using CytoFLEX S (Beckman Coulter, Tokyo, Japan). Live cells were gated using Zombie Green (BioLegend, San Diego, CA, USA). Data were analyzed using the FlowJo software program (version 10.9; Treestar, Ashland, OR, USA). For FACS, stained cells were stored overnight at 4°C with a cell cover (Anacyte Laboratories, Hamburg, Germany). CD11b + /CD45 low cells were considered microglia, and CD11b + /CD45 high cells were considered macrophages. These cells were sorted in ice-cold phosphate-buffered saline (PBS) using a FACS Aria III (BD Biosciences, Franklin Lakes, NJ, USA) with an 85-µm nozzle and the BD FACSDIVA software program (BD Biosciences). Quantitative reverse transcription polymerase chain reaction (RT-PCR) Total RNA from cells, brain tissues, and primary cultured microglia or macrophages was isolated using the Maxwell RSC simplyRNA Tissue/Cells Kit (Promega, Madison, WI, USA). RNA was extracted from sorted microglia and macrophages using an RNeasy Micro Kit (QIAGEN, Valencia, CA, USA). cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix with a gDNA remover kit (Toyobo, Osaka, Japan). qPCR was conducted in triplicate using an MJ Mini instrument (Bio-Rad, Hercules, CA, USA) with THUNDERBIRD™ Next SYBR® qPCR Mix (Toyobo). All PCR primer sequences are listed in Supplementary Table 2 (Hokkaido System Science Co. LTD, Sapporo, Japan). The quantitative PCR (qPCR) data are presented as the percentage of GAPDH mRNA expression levels, calculated as 100 × 1/2 (Ct of target gene – Ct of GAPDH gene) 13 . RNA sequencing (RNAseq) and analyses Total RNA from the brain tissue at the lesion area was isolated as described above. RNA integrity score (RIN) was calculated using the TapeStation system (Agilent Technologies, Santa Clara, CA, USA). Samples with an RIN value above 9.7 were used for further analyses. mRNA purification was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module. Library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit. Sequencing was carried out on a NovaSeq 6000 (Illumina, San Diego, CA, USA) in paired-end 2 × 150-bp cycle mode, generating a total of 26.7 million reads per sample. Data analyses were performed using the CLC Genomics Workbench Premium (QIAGEN) with default settings. In brief, fastq files were trimmed to remove adapter sequences and low-quality bases, followed by mapping and detection of differentially expressed genes (DEGs). Finally, an ingenuity pathway analysis (IPA) (QIAGEN) was used to identify the signaling pathways underlying the transcript sets that significantly predicted the effect of SKF. Detection of superoxide anions using dihydroethidium (DHE) DHE (10 mg/kg) was administered intraperitoneally to SW-TBI model rats 1-day post-injury (dpi) 7 . Thirty minutes later, the animals were euthanized and their brains dissected. For FCM analyses, the tissue from the lesion area was processed into single cells and subjected to magnetic-activated cell sorting using CD11b microbeads to isolate myeloid cells, including microglia and macrophages. After sorting, the cells were analyzed using a violet laser and the V450 channel of CytoFLEX S, and the data were analyzed using the FlowJo software program. Evaluation of oxidative stress using 8-hydroxy-2′-deoxyguanosine (8-OHdG) Brain tissue from the lesion area was isolated at 1 dpi, and DNA was extracted from the tissue using a DNA extraction TIS kit (Wako) and prepared for an enzyme-linked immunosorbent assay (ELISA) using the 8-OHdG Assay Preparation Reagent Set (Wako) 7 . The levels of 8-OHdG were quantified using a high-sensitivity ELISA kit (Nikken SEIL, Shizuoka, Japan). ELISA details Brain tissue from the injury site was harvested and homogenized in RIPA buffer (Wako). TNFα and IL-1β levels were measured using ELISA kits (Elabscience, Houston, TX, USA) and assayed according to the manufacturer's instructions. The protein content of the brain tissue was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) 32 . Immunoblotting For the in vitro study, nuclear fractions of primary microglial cells were collected using a nuclear extraction kit (Active Motif, Tokyo, Japan) 13 . The nuclear fractions were lysed with RIPA buffer, the amount of protein in each sample was quantified with a Pierce BCA Protein Assay Kit, and equal amounts of total protein were prepared with Laemmli sample buffer. For the in vivo study, an equal amount of brain tissue at the lesion area was homogenized in sample buffer. The prepared samples were subjected to immunoblotting using the antibodies listed in Supplementary Table 3. Immunohistochemistry Under deep anesthesia, SW-TBI rats at 1 dpi were subjected to perfusion fixation using 4% paraformaldehyde (PFA; Wako, Osaka, Japan), as described elsewhere 7 . The brain region containing the injury was coronally cryosectioned at a thickness of 16 µm and subjected to immunohistochemical staining using the antibodies listed in Supplementary Table 4. Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. Following immunohistochemical staining with antibodies, the specimens were observed under a FLUOVIEW FV4000 confocal laser scanning microscope (Olympus, Tokyo, Japan). For DHE staining, the dissected brains were immersed in 4% PFA for 30 min and 15% sucrose for 2 h, and then 10-µm-thick frozen sections were prepared and immunostained. Measurement of cellular bioenergetics Quantification of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in rat primary microglial cells and peritoneal macrophages was performed using an XFp Extracellular Flux Analyzer (Agilent Seahorse Bioscience, Santa Clara, CA). After overnight incubation of cells with or without SKF, the plates were placed in the XFp analyzer, which was operated according to the manufacturer’s instructions using oligomycin A (1 µM), carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP; 1 µM), rotenone (0.5 µM), and antimycin A (0.5 M), which were automatically and sequentially added to cells to determine mitochondrial and glycolytic activities 7 . Measurements of lost brain tissue volume Injured rat brains were dissected 9 weeks after SW-TBI and immersed in 4% PFA (Wako) for 1 week, and then six 2-mm-thick slices from the cerebrum centered on the injury site were cut from each brain and photographed. The images were processed to black (cavity) and white (remaining brain tissue) using Adobe Photoshop CS5 Extended (Adobe Systems, San Jose, CA, USA). The percentage of lost volume was calculated 49 . Behavioral assessments Using a video camera and tracking system (Ethovision XT 14; Noldus Info. Tech., Wageningen, Netherlands), the behaviors of SW-TBI and normal control rats were evaluated. In one series of rats, motor incoordination was evaluated with a 5-minute open-field test (OFT) using a square box (100 × 100 cm) with 50-cm-high walls 50 . From the day after the OFT, the Morris water maze (MWM) test was used to evaluate cognitive dysfunction using a 150-cm-diameter × 45-cm-tall circular pool 7 . Statistical analyses Data are expressed as the mean ± standard deviation (SD). Experimental data were analyzed using a two-tailed unpaired t-test, χ 2 test, Fisher’s exact test, or one-way analysis of variance (ANOVA) with Tukey’s post hoc test (the method used in each experiment is described in the figure legends). All analyses were performed using the Prism 9 software program (GraphPad Software, La Jolla, CA, USA). A p-value less than 0.05 was considered significant for all tests. *, **, ***, and **** indicate statistical significance at P < 0.05, 0.01, 0.001, and 0.0001, respectively. Declarations Ethics statement The animal experiments were approved by the Animal Experiment Committee of Ehime University (Approval No. #05U50-2). All methods are reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations. Consent for publication Not applicable. Competing interests The authors have no competing interests. Funding This work was supported by grants from Grant-in-Aid for Scientific Research (C) to [24K12069 (MEC), 23K08383 (TN), and 23K08333 (NA)], Grant-in-Aid for Early-Career Scientists to [21K15699 (NM)], and Grant-in-Aid for Research Activity Start-up [23K19660 (SM)] from the Japan Society for the Promotion of Science (JSPS). Author Contribution Conceptualization: MEC, JT; Formal Analysis: JT; Funding acquisition: MEC, NM, NA, TN, SM; Investigation: MEC, AT, MS, HY(Yamamoto), HY (Yamauchi), KS; Methodology: MEC, NA; Project administration: MEC, JT; Resources: MN, TN, TK; Supervision: TN; Validation: MN, TN, TK; Visualization: MEC, JT; Writing – original draft: MEC, JT; Writing – review & editing: MEC, JT Acknowledgement The authors thankful to Dr. Naohito Tokunaga and Ms. Mei Miyazaki, Bioinformatics Support Division, Advanced Research Support Center (ADRES), Ehime University for assisting RNAseq data analysis, Ms. Makiko Takahashi, Imaging Analysis Support Division, ADRES, Ehime University for helping with brain tissue sectioning, and also to Dr. Yuki Tanaka, Infectious Disease Research Support Division ADRES, Ehime University for flowcytometry analysis and cell sorting. Data Availability The RNA-seq datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository, https://www.ncbi.nlm.nih.gov/geo/, and GEO accession number: GSE298938. The other datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request. References Corps, K. N., Roth, T. L. & McGavern, D. B. Inflammation and neuroprotection in traumatic brain injury. JAMA Neurol. 72 , 355–362 (2015). Gyoneva, S. & Ransohoff, R. M. Inflammatory reaction after traumatic brain injury: therapeutic potential of targeting cell-cell communication by chemokines. Trends Pharmacol. Sci. 36 , 471–480 (2015). Maas, A. I. R. et al. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. Lancet Neurol. 16 , 987–1048 (2017). Maas, A. I. R. et al. Traumatic brain injury: progress and challenges in prevention, clinical care, and research. 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Effects of hypnotic bromovalerylurea on microglial BV2 cells. J. Pharmacol. Sci. 134 , 116–123 (2017). Mori, K. et al. Effects of norepinephrine on rat cultured microglial cells that express alpha1, alpha2, beta1 and beta2 adrenergic receptors. Neuropharmacology 43 , 1026–1034 (2002). Zhang, B. et al. Suppressive effects of phosphodiesterase type IV inhibitors on rat cultured microglial cells: comparison with other types of cAMP-elevating agents. Neuropharmacology 42 , 262–269 (2002). Nishikawa, Y. et al. Anti-inflammatory effects of dopamine on microglia and a D1 receptor agonist ameliorates neuroinflammation of the brain in a rat delirium model. Neurochem Int. 163 , 105479 (2023). Ishii, Y. et al. Anti-inflammatory effects of noradrenaline on LPS-treated microglial cells: Suppression of NFkappaB nuclear translocation and subsequent STAT1 phosphorylation. Neurochem Int. 90 , 56–66 (2015). Tanaka, K. et al. A dopamine D1-like receptor-specific agonist improves the survival of septic mice. iScience 27 , 109587 (2024). Beaulieu, J. M. & Gainetdinov, R. R. The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol. Rev. 63 , 182–217 (2011). Gloire, G., Legrand-Poels, S. & Piette, J. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem. Pharmacol. 72 , 1493–1505 (2006). Morgan, M. J. & Liu, Z. G. Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell. Res. 21 , 103–115 (2011). Brown, G. C. & Neher, J. J. Microglial phagocytosis of live neurons. Nat. Rev. Neurosci. 15 , 209–216 (2014). Sugimoto, K. et al. Activated microglia in a rat stroke model express NG2 proteoglycan in peri-infarct tissue through the involvement of TGF-beta1. Glia 62 , 185–198 (2014). Matsumoto, S. et al. CD200 + and CD200- macrophages accumulated in ischemic lesions of rat brain: the two populations cannot be classified as either M1 or M2 macrophages. J. Neuroimmunol. 282 , 7–20 (2015). Montivero, A. J. et al. Early IGF-1 Gene Therapy Prevented Oxidative Stress and Cognitive Deficits Induced by Traumatic Brain Injury. Front. Pharmacol. 12 , 672392 (2021). Qian, L. et al. beta2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway. J. Immunol. 186 , 4443–4454 (2011). Rasmussen, S. G. et al. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477 , 549–555 (2011). Masel, B. E. & DeWitt, D. S. Traumatic brain injury: a disease process, not an event. J. Neurotrauma . 27 , 1529–1540 (2010). Tu, D. et al. Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration. Free Radic Biol. Med. 200 , 47–58 (2023). Patel, A. R., Ritzel, R., McCullough, L. D. & Liu, F. Microglia and ischemic stroke: a double-edged sword. Int. J. Physiol. Pathophysiol Pharmacol. 5 , 73–90 (2013). Aoto, M. et al. The splicing factor Acin1 is essential for embryonic development but has limited effects on muscle structure and homeostasis. Sci. Rep. 15 , 14017 (2025). Mikami, K. et al. Third-party punishment-like behavior in a rat model. Sci. Rep. 14 , 22310 (2024). Tanaka, J. et al. Induction of resting microglia in culture medium devoid of glycine and serine. Glia 24 , 198–215 (1998). Kikuchi, S. et al. The ameliorative effects of a hypnotic bromvalerylurea in sepsis. Biochem. Biophys. Res. Commun. 459 , 319–326 (2015). Nishihara, T. et al. Subcutaneous injection containing IL-3 and GM-CSF ameliorates stab wound-induced brain injury in rats. Exp. Neurol. 229 , 507–516 (2011). Miyanishi, K. et al. Behavioral tests predicting striatal dopamine level in a rat hemi-Parkinson's disease model. Neurochem Int. 122 , 38–46 (2019). Supplementary Files 250507TBISKFsupplementarytable1.pdf 250507TBISKFsupplementarytable2.pdf 250507TBISKFsupplementarytable3.pdf 250507TBISKFsupplementarytable4.pdf 250507TBISKFsupplementaryfigures.docx Cite Share Download PDF Status: Posted Version 1 posted 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-6726043","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":491635911,"identity":"0a42b03b-0f99-4e99-a4ed-751697b50626","order_by":0,"name":"Mohammed E. Choudhury","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYHACNiBmTuAHkQwGKKIEtEg2kKzF4ABICzFAt7352YOPbdZ5xtcOMD4uKNgmxyCRwPjhBwNfHi4tZmeOmRvObEsvNrudwGw8w+C2MVALs2QPA1sxTi03ctikedsOJ267ncAmzWNwO3H/jQQGaaBzExtwabn/BqJl8+wE9t9ALfUNQFt+49VygweiZYN0AhszUEsC0GFs+G05k2YmOeNceuKM24nNIIcZNvA8bLPsMcDjl+OHn0l8KLNO7J+dfPAzz5/b8gzsyYdv/Kg4hjPEkABjAxLD4FgCEVpQQQ3pWkbBKBgFo2C4AgDq7VGxDRv1WAAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Mohammed","middleName":"E.","lastName":"Choudhury","suffix":""}],"badges":[],"createdAt":"2025-05-22 14:38:23","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6726043/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6726043/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87706329,"identity":"21111b20-c278-450a-9b23-9f5c6ee3fb8e","added_by":"auto","created_at":"2025-07-28 08:06:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":72914,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of DA receptors in the brain and microglia/macrophages. A) Expression of mRNA encoding D1-like receptors, D1R and D5R in normal healthy control (CNT) rat brains, vehicle (Vcl)- and SKF-treated SW-TBI rat brains. A one-way ANOVA and Tukey’s multiple comparison test. ***, **** indicate statistical significance at p \u0026lt;0.001 and 0.0001, respectively. ns, not significant. B) By flow cytometry analysis, microglia and macrophages isolated from the injured brain tissues were distinguished by CD45 expression level. C) Expression of DA receptors by microglia (Ca) and macrophages (Cb). Expression of mRNA encoding D1R, D3R, and D5R was detected in both types of cells. \u003cem\u003en\u003c/em\u003e= 4.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/3e514e057fb34a8bf9875899.png"},{"id":87708143,"identity":"a58d4440-1aca-4dae-b42b-34d7d9073822","added_by":"auto","created_at":"2025-07-28 08:14:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":216347,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SKF on the outcome of SW-TBI. A) Representative brain slices of Vcl- and SKF-treated rats (Aa) prepared two months after SW-TBI. SKF reduced SW-TBI-caused brain tissue loss. Lost brain tissue volumes were statistically analyzed by comparing the ratio (%) of the right (lesioned side) to left (control side) areas. \u003cem\u003en \u003c/em\u003e= 10. Unpaired two-tailed t test. B) Open-field test. (Ba) Representative heat maps of tracked rats’ moving in 5 min. (Bb) Total moved distance in 5 min in OFT. \u003cem\u003en \u003c/em\u003e= 13 (CNT), 15 (Vcl), and 16 (SKF). \u003cstrong\u003eC\u003c/strong\u003e) Morris water maze (MWM) test. (Ca) Representative heat maps of MWM showing the movements of a CNT, a Vcl, and a SKF-treated rat. The position where the platform was placed the previous day was denoted with an arrow, and the target quadrant or nearby zone with an asterisk. (Cb) Total moved distance during the 5-min MWM test. (Cc) Frequency entering the nearby zone. \u003cem\u003en\u003c/em\u003e = 8 (CNT), 10 (Vcl), and 11 (SKF). A one-way ANOVA and Tukey’s multiple comparison test. *, **, ***, **** indicate statistical significance at p \u0026lt;0.05, 0.01, 0.001, and 0.0001, respectively.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/47fb6b5f87314b4b1df9e108.png"},{"id":87708132,"identity":"8d92a54e-344b-4587-8e76-51229f686b2d","added_by":"auto","created_at":"2025-07-28 08:14:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136410,"visible":true,"origin":"","legend":"\u003cp\u003eSuppressive effects of SKF on proinflammatory changes in injured brain tissues. A) IL-1b and TNFa mRNA expression was increased in the injured tissues, and SKF suppressed their expression significantly at 1 dpi as determined by qPCR. \u003cem\u003en\u003c/em\u003e = 8. B) IL-1b and TNFa protein expression was increased in the injured tissues, and SKF suppressed their expression significantly according to an ELISA. \u003cem\u003en\u003c/em\u003e = 8. C) FCM analyses revealed increased infiltration of CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003ehigh\u003c/sup\u003e macrophages and granulocytes, and decreased percentage of CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003elow\u003c/sup\u003e microglia in number. \u003cem\u003en\u003c/em\u003e = 6. D) FCM analyses revealed increased CD11b expression in injured tissues by macrophages. SKF suppressed CD11b expression by microglia and macrophages. \u003cem\u003en\u003c/em\u003e = 6. E) SW-TBI increased CD45 expression by microglia but not macrophages, a finding that was partially reversed by SKF. F) Similarly to CD45, FS values were increased only in microglia, and SKF suppressed the increase. \u003cem\u003en \u003c/em\u003e= 6. G) Microglia were sorted from the injured tissues, and their mRNA expression was investigated with qPCR. SKF decreased the expression of mRNA encoding IL-1b, TNFa, andCD86 in sorted microglia. \u003cem\u003en \u003c/em\u003e= 3. H) SKF did not change the expression of IL-1b, TNFa, and CD86 mRNA in sorted macrophages. \u003cem\u003en \u003c/em\u003e= 3. A one-way ANOVA with Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/f52ff098b1e1920c7602b9e7.png"},{"id":87708647,"identity":"ab6794db-9198-45db-bf54-8472fdfa1865","added_by":"auto","created_at":"2025-07-28 08:22:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":378091,"visible":true,"origin":"","legend":"\u003cp\u003eSKF reduced oxidative stress in the injured tissues. A) 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was increased in the amounts in the injured tissues as revealed by an ELISA, while SKF suppressed the increase. \u003cem\u003en\u003c/em\u003e = 6. B) Fluorescence intensity of dihydroethidium (DHE) reflecting from ROS generation by CD11b\u003csup\u003e+\u003c/sup\u003e cells isolated from SKF-treated brains was weaker than that by the cells from Vcl-treated brains. \u003cem\u003en\u003c/em\u003e = 6. C) Immunohistochemical identification of cells bearing DHE-derived red fluorescence. The strong red fluorescence was found in CD45\u003csup\u003e-\u003c/sup\u003e/Iba1\u003csup\u003e-\u003c/sup\u003e cells (arrowheads). SKF reduced the fluorescence bearing cells. D) mRNA encoding p47phox, p22phox, and gp91phox was increased in the injured tissues and SKF decreased the expression. \u003cem\u003en\u003c/em\u003e = 7. E) Expression of mRNA encoding anti-oxidative enzymes GPX4, CuZnSOD, and catalase was increased in the injured tissues and the increased was reversed by SKF. \u003cem\u003en\u003c/em\u003e = 7. F) SKF prevented the accumulation of NFkB in the nuclei of LPS-treated peritoneal macrophages as revealed by immunoblotting of nuclear fractions. Representative blots and statistical analyses on \u003cem\u003en\u003c/em\u003e = 3 data are shown. G) Phosphorylated IkB kinase (pIKK) was detected in the injured tissues and the ratio of pIKK-immunoreactivity to IKK-immunoreactivity in immunoblotting was reduced by SKF. Representative blots and statistical analysis on \u003cem\u003en\u003c/em\u003e = 5 data are shown. H) Immunohistochemically stained tissues around the injured cores using antibodies to pIKK, CD11b, and NeuN. pIKK\u003csup\u003e+\u003c/sup\u003e cells were frequently present in the Vcl-treated brain (Ha) but such cells were much less in the SKF-treated brain (Hb).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/03cee001d3f6f56522cf95f8.png"},{"id":87706335,"identity":"2d281912-23f2-4ff0-8d29-f598acb902f9","added_by":"auto","created_at":"2025-07-28 08:06:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":158833,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SKF on expression of mRNA encoding CD68, C1qb, TGFb1, and IGF1 in injured brain tissues (A), cultured microglial cells (B), and cultured peritoneal macrophages. A) cDNA for qPCR was prepared from the injured tissues of control and Vcl-treated and SKF-treated rats. B) SW-TBI increased bFGF and TGFb1 expression, and SKF did not exert apparent effects. SW-TBI and SKF did not affect the expression of IGF1 in the injured tissues. \u003cem\u003en\u003c/em\u003e = 8. B) Effects of LPS and SKF on RNA expression by cultured microglia \u003cem\u003en\u003c/em\u003e = 3. D) Effects of LPS and SKF on RNA expression by cultured macrophages \u003cem\u003en\u003c/em\u003e = 3.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/1a9c8c09900b644e66b22db9.png"},{"id":87709971,"identity":"bfbdc097-9873-4ba1-899e-7f7a046d177e","added_by":"auto","created_at":"2025-07-28 08:30:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":464381,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of SKF on comprehensive gene expression. A) Venn diagram of RNAseq results. A total of 7415 genes had variable expression compared to normal subjects by SW-TBI, 72 of which were affected by SKF. \u003cem\u003en\u003c/em\u003e = 8. B) A gene expression network analysis, showing SKF-induced changes in gene expression, mostly of pro-inflammatory factors such as IL-1b. \u003cem\u003en\u003c/em\u003e = 8. C) Volcano plot showing that SKF treatment decreased the expression of many proinflammatory factors, such as IL-1b, IL-1a, and NOS2. \u003cem\u003en\u003c/em\u003e = 8.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/030e3a441727c139da1905a8.png"},{"id":87709969,"identity":"60c536fe-eb56-4698-acbe-5a468632c52d","added_by":"auto","created_at":"2025-07-28 08:30:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":121865,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of an adrenergic β2AR agonist, clenbuterol (CLB). A) The effect of CLB on motor activity of SW-TBI rats. CLB improved moved distance of rats in OFT. \u003cem\u003en \u003c/em\u003e= 7. B) The effects of CLB on accumulation of leukocytes in the injured brains. CLB also showed an inhibitory effect on the accumulation of macrophages and granulocytes. \u003cem\u003en \u003c/em\u003e= 6. C, D) Similar to SKF, CLB also showed suppression of CD11b (C) and CD45 (D) expression in microglia but not in macrophages around the injured tissue. \u003cem\u003en \u003c/em\u003e= 6. E) The results of qPCR examining gene expression around the injured tissue. Like SKF, CLB suppressed the expression of IL-1b, TNFa, and p22phox, and did not affect the expression of Cu/ZnSOD. \u003cem\u003en \u003c/em\u003e= 7.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/0a8a231a0f5732d2baadd649.png"},{"id":87706346,"identity":"d3836db4-ec6a-424f-b470-f1138d6a0f7e","added_by":"auto","created_at":"2025-07-28 08:06:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":347194,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract: SKF-81297 exerts its anti-inflammatory effects by increasing cAMP levels and inhibiting NF-κB nuclear translocation, and also directly inhibits ROS-induced NF-κB activation.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/0c2f0d1d725c677013d493fa.png"},{"id":88505216,"identity":"f3ce43e3-8194-4da3-bc2b-b663dae96cd3","added_by":"auto","created_at":"2025-08-07 07:21:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2830688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/7cbd2080-d4c5-421d-bebd-d7d944e330e1.pdf"},{"id":87708133,"identity":"4f021fe1-c59b-490d-aca2-212a3502ddf2","added_by":"auto","created_at":"2025-07-28 08:14:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15268,"visible":true,"origin":"","legend":"","description":"","filename":"250507TBISKFsupplementarytable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/ebbf977bc1939a394015a0ac.pdf"},{"id":87706334,"identity":"e5384715-5a19-4254-b86f-699e8dfc9f8a","added_by":"auto","created_at":"2025-07-28 08:06:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18837,"visible":true,"origin":"","legend":"","description":"","filename":"250507TBISKFsupplementarytable2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/378acfe45fc752239d4f1dd1.pdf"},{"id":87706337,"identity":"7720eb3b-2851-4d9a-be9c-84b0ceb39332","added_by":"auto","created_at":"2025-07-28 08:06:20","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20102,"visible":true,"origin":"","legend":"","description":"","filename":"250507TBISKFsupplementarytable3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/e0935aa12190146c2a40649e.pdf"},{"id":87708649,"identity":"fe755137-c882-4876-93a5-e78775f9252b","added_by":"auto","created_at":"2025-07-28 08:22:20","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15875,"visible":true,"origin":"","legend":"","description":"","filename":"250507TBISKFsupplementarytable4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/6f890d7b97f809eb23ce1368.pdf"},{"id":87708652,"identity":"7a1b6eb5-8388-469a-94e6-af68da1df335","added_by":"auto","created_at":"2025-07-28 08:22:20","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":942001,"visible":true,"origin":"","legend":"","description":"","filename":"250507TBISKFsupplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6726043/v1/6b1d01a82f4be803b552e81f.docx"}],"financialInterests":"","formattedTitle":"A dopamine D1-like receptor agonist ameliorates stab wound-induced brain injury through its immunosuppressive effect","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eMicroglia and macrophages in stab wound-induced traumatic brain injury (SW-TBI) expressed dopamine receptors.\u003c/li\u003e\n \u003cli\u003eA dopamine D1-like receptor agonist ameliorated SW-TBI-induced behavioral deficits.\u003c/li\u003e\n \u003cli\u003eThe agonist inhibited cellular ROS generation and NF\u0026kappa;B nuclear translocation.\u003c/li\u003e\n \u003cli\u003eGs-linked GPCRs mediate suppressive effects on TBI-induced neuroinflammation.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eTraumatic brain injury (TBI) is a leading cause of severe brain dysfunction, including motor, sensory, mental, and cognitive deficits, as well as acute mortality \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Even more worrying is the increasing number of stab wound-induced traumatic brain injury (SW-TBI) caused by knives and firearms, which directly damage blood vessels and brain tissue, causing intracerebral hemorrhages \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Numerous studies have demonstrated that chronic brain dysfunction resulting from TBI is closely associated with neuroinflammation, secondary to TBI-induced primary neural cell death. In this secondary neuroinflammation, resident microglia and infiltrating leukocytes (monocytes, granulocytes, and lymphocytes) play crucial roles in exacerbating pathophysiological processes in and around the injured tissues \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These immune cells release various proinflammatory mediators, including cytokines, reactive oxygen species (ROS), and nitric oxide. Monocyte-derived macrophages and activated microglia also accelerate neural cell death through phagocytic activity that is a process known as phagoptosis \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn addition to their detrimental roles, microglia and circulating monocyte-derived macrophages may contribute to preventing degeneration and promoting the regeneration of injured tissues by eliminating degenerated materials and releasing anti-inflammatory cytokines and neuroprotective growth factors \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Furthermore, these cells have the potential to scavenge ROS while suppressing ROS-induced cell death, including ferroptosis \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Single-cell RNA-sequencing data from a recent report using stab wound brain injury showed striking and distinct transcriptional changes in glial cell populations\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, interventions that suppress the detrimental nature and promote the beneficial functions of these immune cells accumulating in the injured tissues are of great interest.\u003c/p\u003e\u003cp\u003eTo date, various pharmacological interventions for TBI have been explored to suppress the detrimental effects and promote the beneficial functions of immune cells, particularly microglia and macrophages \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Methylprednisolone, a corticosteroid studied for its potential to reduce inflammation TBI, has been administered to patients with SW-TBI \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. N-acetylcysteine, an antioxidant that may help reduce oxidative stress, has been investigated for its ameliorative effects in both laboratory \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and clinical settings \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In our previous study, classical hypnotic bromvalerylurea (BU), which has anti-inflammatory effects on microglia and macrophages in addition to enhancing GABAA receptor-mediated neurotransmission, markedly improved outcomes in rats with SW-TBI \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. BU exerts anti-inflammatory effects by activating Nrf2 [12] and by inhibiting JAK1\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAgents that increase intracellular cAMP levels have been reported to suppress the proinflammatory activation of microglia \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In particular, β2 adrenergic receptor (AR) agonists have strong immunosuppressive effects on lipopolysaccharide (LPS)-induced proinflammatory activation of microglia \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Microglia also express another G protein-coupled receptor (GPCR), the dopamine D1-like receptor, which is linked to Gs proteins \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Dopamine and the D1-like receptor-specific agonist SKF-81297 (SKF) suppress LPS-induced IL-1b and TNFa expression while increasing cAMP levels in primary rat microglia \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven the strong immunosuppressive effects of SKF, we investigated whether or not SKF ameliorates SW-TBI in a rat model by suppressing the proinflammatory activities of microglia and infiltrating leukocytes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExpression of dopamine receptors\u003c/h2\u003e\u003cp\u003eDopamine receptors D1 (DR1) and DR5 are classified as D1-like receptors, which are GPCR linked to G protein Gs. mRNA encoding DR1 and DR5 was expressed in the striatum of control, Vcl-, and SKF-81297 (SKF)-injected SW-TBI rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The D1-like receptor-specific agonist SKF suppressed LPS-induced IL-1b and TNFa expression in primary rat microglia and peritoneal macrophages (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe injured core and its surrounding tissue were enzymatically dispersed in single cells, which were subjected to an FCM analysis using antibodies against CD11b and CD45 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Infiltrated macrophages were identified as CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003ehigh\u003c/sup\u003e cells and resident microglia as CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003elow\u003c/sup\u003e cells. Both cell lines were sorted to prepare the total RNA for qPCR. The expression of mRNA encoding DR1, DR3, and DR5 was detected in both sorted microglia and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAmeliorative effects of SKF on SW-TBI\u003c/h3\u003e\n\u003cp\u003eSKF was administered to SW-TBI rats once daily for 7 days, and its ameliorative effects were evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Probably due to secondary degeneration \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, SW-TBI generates severe brain tissue loss on a monthly basis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. SKF suppressed brain tissue loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The total distance moved in the open-field test (OFT) decreased in SW-TBI rats, but SKF reversed this reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). SW-TBI has been shown to disturb cognitive and learning/memory functions \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The MWM test showed that SW-TBI did not cause apparent changes in the total distance moved, but SKF increased the frequency of entering the target quadrant in the pool, where the platform was placed under the waterline on the previous day (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This suggests that SW-TBI-induced cognitive and learning abilities were ameliorated by SKF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffects of SKF on proinflammatory responses\u003c/h3\u003e\n\u003cp\u003e\u003c/p\u003e\u003cp\u003eSW-TBI increased expression of IL-1b and TNFa at mRNA and protein levels in and around the injured core (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, respectively). SKF suppressed SW-TBI-induced elevated expression of these cytokines. FCM analyses showed that the percentage of total CD11b\u003csup\u003e+\u003c/sup\u003e cells decreased in injured brain tissues, while macrophages and granulocytes increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). SKF reversed SW-TBI-induced changes in the CD11b\u003csup\u003e+\u003c/sup\u003e cell population. The microglia and macrophages were further characterized using FCM. SKF decreased CD11b expression in both microglia and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), whereas CD45 expression decreased in microglia but not in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Forward scatter (FS) values also decreased only in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Microglia and macrophages were sorted from injured tissues at 1 dpi, and total RNA was isolated for qPCR. SKF administration decreased the mRNA expression of proinflammatory mediators IL-1b, TNFa, and CD86 in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) but not in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e\n\u003ch3\u003eSuppression of NFkB-mediated proinflammatory pathways by SKF\u003c/h3\u003e\n\u003cp\u003eROS cause the generation of 8-OHdG, a marker of oxidative damage to DNA. To determine whether or not SKF reduced oxidative stress in SW-TBI tissues, 8-OHdG levels in injured tissues dissected at 1 dpi were measured by an ELISA. SW-TBI significantly increased 8-OHdG levels, which were reduced by SKF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). DHE is oxidized to emit red fluorescence from ethidium \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. CD11b\u003csup\u003e+\u003c/sup\u003e cells were sorted using MACS and analyzed by flow cytometry to measure oxidized DHE-derived fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The mean fluorescence intensity was reduced in CD11b\u003csup\u003e+\u003c/sup\u003e cells sorted from SKF-treated rat brains compared to vehicle (Vcl)-treated rat brains. However, SKF-induced reduction of DHE-derived fluorescence by CD11b\u003csup\u003e+\u003c/sup\u003e cells was not marked, suggesting that CD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e cells may be responsible for ROS generation.\u003c/p\u003e\u003cp\u003eAn immunohistochemical analysis showed that most Iba1\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e ramified microglial cells did not exhibit red fluorescence, and Iba1\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003e+\u003c/sup\u003e round macrophages were weakly positive for DHE-derived fluorescence (Fig.\u0026nbsp;4Ca, Supplementary Fig.\u0026nbsp;2A). Furthermore, the study suggests that Iba1\u003csup\u003e\u0026minus;\u003c/sup\u003e/CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e cells are the major source of ROS. Compared to Vcl-treated brain sections, SKF-treated sections displayed much weaker red fluorescence (Fig.\u0026nbsp;4Cb), suggesting that SKF might have suppressed ROS generation by not only myeloid cells but also neuroectodermal cells, such as neurons \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo reveal the mechanisms by which SKF reduced oxidative injury, the expression of oxidative and anti-oxidative enzymes was investigated by qPCR (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The expression of gp91phox, a subunit of NADPH oxidase 2 (NOX2), was upregulated in Vcl-treated injured tissues, and this upregulation was reversed by SKF administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The expression of p47phox and p22phox, subunits necessary for NOX2 activity, was also increased by SW-TBI and suppressed by SKF. Conversely, the expression of antioxidative enzymes glutathione peroxidase 4 (GPX4), Cu/Zn superoxide dismutase (Cu/ZnSOD), and catalase was not elevated in injured tissues, and SKF did not affect (GPX4) or decrease the expression of Cu/ZnSOD and catalase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eROS have been shown to induce NFκB activation, a critical transcription factor that elevates the expression of various proinflammatory factors, such as IL-1b and TNFa \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Immunoblotting revealed that LPS induced NFκB p65 localization in the nuclear fraction of rat peritoneal macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). SKF prevented LPS-induced nuclear localization of p65. Phosphorylated IkB kinase (pIKK) induces the degradation of IkB, resulting in nuclear localization of p65 \u003csup\u003e31\u003c/sup\u003e. To examine whether or not SKF suppressed the SW-TBI-induced nuclear localization of p65 in injured brain tissues, immunoblotting was performed to detect pIKK in injured tissues. SW-TBI caused phosphorylation of IKK in injured tissues, and SKF administration prevented this phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). To determine the cell type bearing pIKK in and around injured tissues, an immunohistochemical study was conducted. Figure\u0026nbsp;4Ha shows that the pIKK\u003csup\u003e+\u003c/sup\u003e/CD11b\u003csup\u003e+\u003c/sup\u003e cells with morphological characteristics of infiltrated macrophages that are a round morphology without apparent ramified processes \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In contrast, ramified CD11b\u003csup\u003e+\u003c/sup\u003e cells or microglia appeared to be pIKK\u003csup\u003e\u0026minus;\u003c/sup\u003e. In SKF-treated rat tissues, the number of pIKK\u003csup\u003e+\u003c/sup\u003e cells was much lower than that in Vcl-treated rat tissues (Fig.\u0026nbsp;4Hb). In addition to microglia/macrophages, an abundant number of CD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e cells appeared to be pIKK\u003csup\u003e+\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;2b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePro-inflammatory responses in immune cells are correlated with metabolic changes. SKF was found to suppress both anaerobic glycolytic and aerobic mitochondrial metabolism, as revealed by a flux analysis (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003ch3\u003eEffects of SKF on anti-inflammatory, phagocytic, and neuroprotective responses\u003c/h3\u003e\n\u003cp\u003eThe phagocytic activity of microglia and macrophages may be responsible for tissue restoration, as injury-induced accumulation of degenerated materials prevents restoration \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Conversely, it has also been proposed that viable cells are killed by the phagocytosis of microglia and macrophages. This aggravating process is called phagocytosis and is mediated by the opsonization of damaged cells with complement C1q \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. SW-TBI increased the expression of CD68, a marker for phagosomes, and complement C1qb, and SKF reduced the increased CD68 and C1qb expression in the injured brain tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). SW-TBI increased the expression of the typical anti-inflammatory cytokine TGFb1 \u003csup\u003e13, 37\u003c/sup\u003e, but SKF did not affect TGFb1 expression in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). IGF1 is a neuroprotective factor involved in the restoration of injured brains, and blood-borne infiltrating macrophages may be a source of IGF1\u003csup\u003e12, 38, 39\u003c/sup\u003e. SW-TBI did not affect IGF1 expression, but SKF tended to reduce IGF1 expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e study showed that SKF did not affect CD68 expression in cultured rat primary microglia or peritoneal macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, SKF reduced C1qb expression in microglia cultured in the presence of LPS. SKF reduced microglial TGFb1 and IGF1 expression in both the absence and presence of LPS. Similar suppressive effects of SKF on TGFb1 and IGF1 expression by macrophages were only observed in the absence of LPS because LPS strongly suppressed TGFb1 and IGF1 expression. These data suggest that the ameliorative effects of SKF are independent of the immunosuppressive cytokine TGFb1 and the neuroprotective factor IGF1.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffects of SKF on comprehensive gene expression in the injured brain tissues.\u003c/em\u003e\u003c/p\u003e\u003cp\u003eRNAseq was conducted to comprehensively investigate gene expression, and the results are presented in a Venn diagram, illustrating the expression changes of 7,343 genes induced by SW-TBI. The expression of 72 genes was altered by SKF treatment. The primary genes with altered expression were a group of proinflammatory mediators, such as IL-1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). A pathway analysis was performed to identify the specific pathways through which SKF exerted its effects. The analysis revealed that SKF inhibited pathways that promote immune cell activation and leukocyte infiltration into the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). A volcano plot demonstrated that the expression of many proinflammatory factors, such as IL-1b and NOS2, was decreased by SKF treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEffects of the b2AR-specific agonist clenbuterol (CLB) on SW-TBI\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effects of CLB, a BBB-permeable b2AR-specific agonist \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, on SW-TBI were investigated. b2AR is a Gs-coupled GPCR identical to dopamine D1-like receptors \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. CLP increased motor activity of SW-TBI rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). CLB had an inhibitory effect on macrophage and granulocyte accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). CLB also suppressed CD11b and CD45 expression in microglia in and around injured tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). A qPCR analysis of gene expression around injured tissues revealed that CLB suppressed the mRNA expression of IL-1b, TNFa, and p22phox but did not affect Cu/ZnSOD expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSW-TBI is still an intractable condition, for which there are still no specific and effective interventions. SW-TBI has a high mortality rate, and most survivors suffer from neurological and psychological sequelae, which often severely disturb their well-being in daily life. Although microglia themselves may not be thought to have strong deleterious effects on the brain \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, they can induce infiltration into the injured tissues of circulating leukocytes, especially monocytes, which are the precursor cells of brain macrophages \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The infiltrated leukocytes, in particular monocyte-derived macrophages, play aggravating effects on injured brains through several mechanisms: releasing pro-inflammatory mediators, such as proinflammatory cytokines/chemokines; generating ROS causing oxidative injuries in brain tissues and cells; and eliminating viable cells by phagocytosis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Thus, microglia are thought to play a central role in the pathogenesis of SW-TBI.\u003c/p\u003e\u003cp\u003eAlthough both microglia and macrophages express D1R and D5R, SKF may act more strongly on microglia than macrophages, and it more markedly suppresses the pro-inflammatory activation of microglia than macrophages \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. SKF reduced the infiltration of monocytes and granulocytes, probably due to the reduced expression of proinflammatory mediators by microglia, resulting in the improvement of SW-TBI pathogenesis. SKF ameliorated the oxidative damage in and around the injured tissue caused by SW-TBI by suppressing the metabolic activity of microglia and macrophages and ameliorating the chronically progressive loss of brain tissue and cognitive dysfunction caused by SW-TBI, as well as motor inactivity. These results strongly suggest that pharmacological intervention to activate D1-like receptor-mediated signaling in SW-TBI pathology may improve the prognosis.\u003c/p\u003e\u003cp\u003eRNAseq results showed that SKF suppressed the expression of proinflammatory mediators, such as IL-1b, which was driven by NFκB \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. SKF may suppress translocation of the proinflammatory transcription factor NFκB into the nuclei to prevent proinflammatory responses in the injured brain. SKF reduced oxidative stress-induced injury, which was likely correlated with decreased proinflammatory reactions. SKF suppresses NOX2 enzyme subunit expression The present immunohistochemical observations suggest that CD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e neural cells may be a critical source of ROS. Neurons express NOX2 during inflammation, chronically causing neurodegeneration \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. SKF may have suppressed neuronal ROS generation. In contrast, SKF did not increase the expression of the anti-oxidative enzymes GPX4 or Cu/ZnSOD. These results suggest that although SKF suppresses NFκB, it is unlikely to increase the activity of the transcription factor NF-E2-related factor 2 (Nrf2), which increases the expression of antioxidant factors and enhances anti-inflammatory effects \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRegarding the mechanism of suppression of NFκB activity by SKF, it is well known that when intracellular cAMP concentration is elevated by b2AR agonists \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e or phosphodiesterase inhibitors \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, nuclear translocation of NFκB is prevented [24]. As both D1-like receptors and b2AR are Gs-coupled GPCRs \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, it is likely that they also inhibit the nuclear translocation of NFκB via an increase in intracellular cAMP concentration. In fact, this study showed that the b2AR agonist CLB exerted anti-inflammatory effects similar to SKF, suggesting that the ameliorating effects of SKF on SW-TBI were mediated by its cAMP-elevating effects on microglia and macrophages \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMicroglia and infiltrating macrophages are not always detrimental to injured brains \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. These cells release several neuroprotective cytokines and growth factors, such as IGF1, BDNF, and bFGF. They also secrete anti-inflammatory cytokines, including TGFβ1, which have strong and sustained effects in suppressing the proinflammatory activation of infiltrating macrophages. Thus, microglia and macrophages are often described as a \"double-edged sword\" \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Given the above, the ameliorative effects of SKF on SW-TBI may be attributed to the enhancement of the neuroprotective and anti-inflammatory actions of microglia and macrophages. However, as shown in this study, SKF decreased TGFβ1 and IGF1 expression. Stimulative effects of SKF on the expression of immunosuppressive or neuroprotective factors were not observed in the RNAseq study. Thus, it is likely that SKF ameliorates the prognosis of SW-TBI by suppressing SW-TBI-induced neuroinflammation through the inhibition of nuclear localization of NFκB. A concise visual representation of the primary findings of the study is presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn conclusion, SKF, a blood-brain barrier-permeable dopamine D1-like receptor agonist, suppresses brain inflammation and improves the functional prognosis in a rat SW-TBI model. These results suggest that D1-like agonists are effective for the treatment of brain injury, a refractory pathology. This action was thought to be associated with an elevation of the intracellular cAMP concentration and appeared through the inhibition of the expression of proinflammatory mediators.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003e All rats received standard care, and our study protocol was approved by Ethics Committee for Animal Experimentation of Ehime University, Japan (approval number #05U50-2). In accordance with the previous reports of our university, all approaches accomplished in this study strictly followed to the ARRIVE guidelines. Our animal facility-bred 10- to 12-week-old male Wistar rats weighing from 250 to 300 g (Clea Japan, Tokyo, Japan) were used in this study. The SW-TBI model was prepared by inducing stab wounds in the rat brain as described elsewhere \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In brief, under deep anesthesia with isoflurane (Mylan Pharmaceutical company, Tokyo, Japan), the rat's head was secured in a stereotaxic apparatus (Narishige, Tokyo, Japan), and a longitudinal incision of approximately 15 mm was made in the skin to expose the skull. Two holes were drilled through the skull over the right hemisphere at approximately 2.5 and 4 mm right of the midline and 1 mm posterior to the bregma, respectively. A 26-gauge needle was inserted through each hole to a depth of approximately 7 mm from the surface of the skull and moved in a fan-like manner from anterior to posterior, parallel to the midline. The needle was then withdrawn, and the skin incision was closed with quick-drying glue (Aron-Alpha; Toagosei, Tokyo, Japan). Soldem 3A solution (10 ml/rat/day; Terumo Corporation, Tokyo, Japan) was administered for postoperative care. For Euthanasia, CO2 (Matsuyama Nishi Sanso Company, Matsuyama, Japan) inhalation exposure was performed where CO₂ flow rate was maintained to replace 30\u0026ndash;50% of the cage volume per minute\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePrimary microglia and macrophage cell cultures\u003c/h2\u003e\u003cp\u003eRat primary microglial cell cultures were prepared as previously described \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, where cortices from newborn rat pups were mechanically dissociated into individual cells. Dissociated cells were cultured as mixed glial cultures in 75 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e flasks with 10% fetal calf serum (FCS)-supplemented Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM; Fujifilm Wako, Osaka, Japan). After 11 days, microglial cells were obtained by agitating the flasks at 200 rpm for 1 h at 37\u0026deg;C, and pure microglial cells were plated in poly L-lysine-coated 6-well culture plates for an additional 11 days. Rat primary macrophages were harvested from the peritoneal lavage of adult rats as described elsewhere \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and cultured in DMEM supplemented with 3% FCS-supplemented DMEM (Fujifilm Wako). For pharmacological studies, the culture medium was removed, and the cells were incubated for 6 hours in serum-free E2 medium (DMEM containing 10 mM HEPES, pH 7.3; Gibco, Grand Island, NY, USA; 4.5 mg/mL glucose, Gibco; 5 \u0026micro;g/mL insulin, 5 nM sodium selenite, 5 \u0026micro;g/mL transferrin, Gibco; and 0.2 mg/mL bovine serum albumin, Sigma-Aldrich, St. Louis, MO, USA). Inflammatory cell culture models were prepared by exposing the cells to E2 medium containing 1 \u0026micro;g/mL LPS (from \u003cem\u003eEscherichia coli\u003c/em\u003e, serotype 055:B5; Sigma-Aldrich) for 6 h or overnight, as required by each assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePharmacological interventions\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eStudies\u003c/b\u003e: The D1-like receptor-selective agonist SKF-81297 hydrobromide (SKF; Tocris Bioscience, Bristol, UK) was dissolved in DMSO and diluted with normal saline to a concentration of 2.5%. Normal saline containing 2.5% DMSO was used as the Vcl. SKF was administered intraperitoneally to SW-TBI rats at a dose of 10 mg/kg, 1 h after SW-TBI. In the long-term experiments, SKF was administered once per day for 7 days at the same dose. Clenbuterol hydrochloride (CLB; Sigma-Aldrich), dissolved in the same manner as SKF, was administered subcutaneously at a dose of 0.1 mg/kg body weight, following the same administration schedule as SKF.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eStudies\u003c/b\u003e: LPS-treated and untreated cells were exposed to 10 \u0026micro;M SKF.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry (FCM) and fluorescence-activated cell sorting (FACS)\u003c/h2\u003e\u003cp\u003eBrain tissue from the lesion area (approximately 100 mg) of SW-TBI and normal rats was dissociated into single cells using a gentleMACS dissociator with 37C_ABDK (Miltenyi Biotec, Tokyo, Japan) and an adult brain dissociation kit (Miltenyi Biotec), as previously described \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The prepared cell suspensions were subjected to FCM analyses and FACS. For multicolor immunofluorescence labeling, the cells were blocked with mouse anti-rat CD32 antibody (Rat BD Fc Block; BD Biosciences, Franklin Lakes, NJ, USA), followed by labeling with fluorescent antibodies against cell surface antigens by incubating the cells for 30 min at 4\u0026deg;C using the antibodies listed in Supplementary Table\u0026nbsp;1. Cells labeled with antibodies for flow cytometry were analyzed using CytoFLEX S (Beckman Coulter, Tokyo, Japan). Live cells were gated using Zombie Green (BioLegend, San Diego, CA, USA). Data were analyzed using the FlowJo software program (version 10.9; Treestar, Ashland, OR, USA). For FACS, stained cells were stored overnight at 4\u0026deg;C with a cell cover (Anacyte Laboratories, Hamburg, Germany). CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003elow\u003c/sup\u003e cells were considered microglia, and CD11b\u003csup\u003e+\u003c/sup\u003e/CD45\u003csup\u003ehigh\u003c/sup\u003e cells were considered macrophages. These cells were sorted in ice-cold phosphate-buffered saline (PBS) using a FACS Aria III (BD Biosciences, Franklin Lakes, NJ, USA) with an 85-\u0026micro;m nozzle and the BD FACSDIVA software program (BD Biosciences).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative reverse transcription polymerase chain reaction (RT-PCR)\u003c/h2\u003e\u003cp\u003eTotal RNA from cells, brain tissues, and primary cultured microglia or macrophages was isolated using the Maxwell RSC simplyRNA Tissue/Cells Kit (Promega, Madison, WI, USA). RNA was extracted from sorted microglia and macrophages using an RNeasy Micro Kit (QIAGEN, Valencia, CA, USA). cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix with a gDNA remover kit (Toyobo, Osaka, Japan). qPCR was conducted in triplicate using an MJ Mini instrument (Bio-Rad, Hercules, CA, USA) with THUNDERBIRD\u0026trade; Next SYBR\u0026reg; qPCR Mix (Toyobo). All PCR primer sequences are listed in Supplementary Table\u0026nbsp;2 (Hokkaido System Science Co. LTD, Sapporo, Japan). The quantitative PCR (qPCR) data are presented as the percentage of GAPDH mRNA expression levels, calculated as 100 \u0026times; 1/2\u003csup\u003e(Ct of target gene \u0026ndash; Ct of GAPDH gene) \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing (RNAseq) and analyses\u003c/h2\u003e\u003cp\u003eTotal RNA from the brain tissue at the lesion area was isolated as described above. RNA integrity score (RIN) was calculated using the TapeStation system (Agilent Technologies, Santa Clara, CA, USA). Samples with an RIN value above 9.7 were used for further analyses. mRNA purification was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module. Library preparation was performed using the NEBNext Ultra II Directional RNA Library Prep Kit. Sequencing was carried out on a NovaSeq 6000 (Illumina, San Diego, CA, USA) in paired-end 2 \u0026times; 150-bp cycle mode, generating a total of 26.7\u0026nbsp;million reads per sample. Data analyses were performed using the CLC Genomics Workbench Premium (QIAGEN) with default settings. In brief, fastq files were trimmed to remove adapter sequences and low-quality bases, followed by mapping and detection of differentially expressed genes (DEGs). Finally, an ingenuity pathway analysis (IPA) (QIAGEN) was used to identify the signaling pathways underlying the transcript sets that significantly predicted the effect of SKF.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eDetection of superoxide anions using dihydroethidium (DHE)\u003c/h2\u003e\u003cp\u003eDHE (10 mg/kg) was administered intraperitoneally to SW-TBI model rats 1-day post-injury (dpi) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Thirty minutes later, the animals were euthanized and their brains dissected. For FCM analyses, the tissue from the lesion area was processed into single cells and subjected to magnetic-activated cell sorting using CD11b microbeads to isolate myeloid cells, including microglia and macrophages. After sorting, the cells were analyzed using a violet laser and the V450 channel of CytoFLEX S, and the data were analyzed using the FlowJo software program.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of oxidative stress using 8-hydroxy-2\u0026prime;-deoxyguanosine (8-OHdG)\u003c/h2\u003e\u003cp\u003eBrain tissue from the lesion area was isolated at 1 dpi, and DNA was extracted from the tissue using a DNA extraction TIS kit (Wako) and prepared for an enzyme-linked immunosorbent assay (ELISA) using the 8-OHdG Assay Preparation Reagent Set (Wako) \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The levels of 8-OHdG were quantified using a high-sensitivity ELISA kit (Nikken SEIL, Shizuoka, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eELISA details\u003c/h2\u003e\u003cp\u003eBrain tissue from the injury site was harvested and homogenized in RIPA buffer (Wako). TNFα and IL-1β levels were measured using ELISA kits (Elabscience, Houston, TX, USA) and assayed according to the manufacturer's instructions. The protein content of the brain tissue was quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblotting\u003c/h2\u003e\u003cp\u003eFor the \u003cem\u003ein vitro\u003c/em\u003e study, nuclear fractions of primary microglial cells were collected using a nuclear extraction kit (Active Motif, Tokyo, Japan) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The nuclear fractions were lysed with RIPA buffer, the amount of protein in each sample was quantified with a Pierce BCA Protein Assay Kit, and equal amounts of total protein were prepared with Laemmli sample buffer. For the \u003cem\u003ein vivo\u003c/em\u003e study, an equal amount of brain tissue at the lesion area was homogenized in sample buffer. The prepared samples were subjected to immunoblotting using the antibodies listed in Supplementary Table\u0026nbsp;3.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eUnder deep anesthesia, SW-TBI rats at 1 dpi were subjected to perfusion fixation using 4% paraformaldehyde (PFA; Wako, Osaka, Japan), as described elsewhere \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The brain region containing the injury was coronally cryosectioned at a thickness of 16 \u0026micro;m and subjected to immunohistochemical staining using the antibodies listed in Supplementary Table\u0026nbsp;4. Hoechst 33342 (Sigma-Aldrich) was used for nuclear staining. Following immunohistochemical staining with antibodies, the specimens were observed under a FLUOVIEW FV4000 confocal laser scanning microscope (Olympus, Tokyo, Japan). For DHE staining, the dissected brains were immersed in 4% PFA for 30 min and 15% sucrose for 2 h, and then 10-\u0026micro;m-thick frozen sections were prepared and immunostained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of cellular bioenergetics\u003c/h2\u003e\u003cp\u003eQuantification of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in rat primary microglial cells and peritoneal macrophages was performed using an XFp Extracellular Flux Analyzer (Agilent Seahorse Bioscience, Santa Clara, CA). After overnight incubation of cells with or without SKF, the plates were placed in the XFp analyzer, which was operated according to the manufacturer\u0026rsquo;s instructions using oligomycin A (1 \u0026micro;M), carbonyl cyanide-\u003cem\u003ep\u003c/em\u003e-trifluoromethoxyphenylhydrazone (FCCP; 1 \u0026micro;M), rotenone (0.5 \u0026micro;M), and antimycin A (0.5 M), which were automatically and sequentially added to cells to determine mitochondrial and glycolytic activities \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eMeasurements of lost brain tissue volume\u003c/h2\u003e\u003cp\u003eInjured rat brains were dissected 9 weeks after SW-TBI and immersed in 4% PFA (Wako) for 1 week, and then six 2-mm-thick slices from the cerebrum centered on the injury site were cut from each brain and photographed. The images were processed to black (cavity) and white (remaining brain tissue) using Adobe Photoshop CS5 Extended (Adobe Systems, San Jose, CA, USA). The percentage of lost volume was calculated \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eBehavioral assessments\u003c/h2\u003e\u003cp\u003eUsing a video camera and tracking system (Ethovision XT 14; Noldus Info. Tech., Wageningen, Netherlands), the behaviors of SW-TBI and normal control rats were evaluated. In one series of rats, motor incoordination was evaluated with a 5-minute open-field test (OFT) using a square box (100 \u0026times; 100 cm) with 50-cm-high walls \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. From the day after the OFT, the Morris water maze (MWM) test was used to evaluate cognitive dysfunction using a 150-cm-diameter \u0026times; 45-cm-tall circular pool \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Experimental data were analyzed using a two-tailed unpaired t-test, χ\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e test, Fisher\u0026rsquo;s exact test, or one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test (the method used in each experiment is described in the figure legends). All analyses were performed using the Prism 9 software program (GraphPad Software, La Jolla, CA, USA). A p-value less than 0.05 was considered significant for all tests. *, **, ***, and **** indicate statistical significance at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 0.01, 0.001, and 0.0001, respectively.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cb\u003eEthics statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003e The animal experiments were approved by the Animal Experiment Committee of Ehime University (Approval No. #05U50-2). All methods are reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. We confirmed that all experiments in this study were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors have no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003e This work was supported by grants from Grant-in-Aid for Scientific Research (C) to [24K12069 (MEC), 23K08383 (TN), and 23K08333 (NA)], Grant-in-Aid for Early-Career Scientists to [21K15699 (NM)], and Grant-in-Aid for Research Activity Start-up [23K19660 (SM)] from the Japan Society for the Promotion of Science (JSPS).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: MEC, JT; Formal Analysis: JT; Funding acquisition: MEC, NM, NA, TN, SM; Investigation: MEC, AT, MS, HY(Yamamoto), HY (Yamauchi), KS; Methodology: MEC, NA; Project administration: MEC, JT; Resources: MN, TN, TK; Supervision: TN; Validation: MN, TN, TK; Visualization: MEC, JT; Writing \u0026ndash; original draft: MEC, JT; Writing \u0026ndash; review \u0026amp; editing: MEC, JT\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thankful to Dr. Naohito Tokunaga and Ms. Mei Miyazaki, Bioinformatics Support Division, Advanced Research Support Center (ADRES), Ehime University for assisting RNAseq data analysis, Ms. Makiko Takahashi, Imaging Analysis Support Division, ADRES, Ehime University for helping with brain tissue sectioning, and also to Dr. Yuki Tanaka, Infectious Disease Research Support Division ADRES, Ehime University for flowcytometry analysis and cell sorting.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe RNA-seq datasets generated and/or analyzed during the current study are available in the Gene Expression Omnibus repository, https://www.ncbi.nlm.nih.gov/geo/, and GEO accession number: GSE298938. The other datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCorps, K. N., Roth, T. L. \u0026amp; McGavern, D. B. Inflammation and neuroprotection in traumatic brain injury. \u003cem\u003eJAMA Neurol.\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, 355\u0026ndash;362 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGyoneva, S. \u0026amp; Ransohoff, R. M. Inflammatory reaction after traumatic brain injury: therapeutic potential of targeting cell-cell communication by chemokines. \u003cem\u003eTrends Pharmacol. Sci.\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e, 471\u0026ndash;480 (2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaas, A. I. R. et al. 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Behavioral tests predicting striatal dopamine level in a rat hemi-Parkinson's disease model. \u003cem\u003eNeurochem Int.\u003c/em\u003e \u003cb\u003e122\u003c/b\u003e, 38\u0026ndash;46 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"Traumatic brain injury, microglia, macrophage, SKF-81297, LPS, NFκB, ROS","lastPublishedDoi":"10.21203/rs.3.rs-6726043/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6726043/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraumatic brain injury (TBI) causes progressive nervous tissue degeneration long after the initial injury due to secondary neuroinflammatory reactions. G protein-coupled dopamine D1-like receptors, which elevate intracellular cAMP levels, have been shown to mediate the suppressive effects on lipopolysaccharide (LPS)-induced proinflammatory activation of microglia and macrophages. The present study investigated whether or not the D1-like receptor-specific agonist SKF-81297 (SKF) administered intraperitoneally once daily for 7 days starting 1 h after TBI could ameliorate TBI in a rat model of stab wounds in the forebrain. SKF reduced the volume of TBI-induced brain tissue loss, increased mobile activity, and ameliorated cognitive dysfunction two months after TBI. A single dose of SKF suppressed the expression of IL-1β and TNFα in brain tissue by reducing oxidative injury 24 h post-TBI. SKF decreased the expression of NADPH oxidase 2 subunits but did not affect antioxidative enzymes. SKF also prevented LPS-induced translocation of NFκB into the nuclei of macrophages. The agonist clenbuterol (CLB) for adrenergic β2 receptor, another Gs-linked GPCR, exerted comparable ameliorative effects in TBI model rats by suppressing neuroinflammation. In summary, SKF may exert anti-inflammatory effects by suppressing the NFκB pathway, similar to CLB, leading to amelioration of TBI-induced brain degeneration.\u003c/p\u003e","manuscriptTitle":"A dopamine D1-like receptor agonist ameliorates stab wound-induced brain injury through its immunosuppressive effect","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-28 08:06:16","doi":"10.21203/rs.3.rs-6726043/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":"d004ca26-7342-4f17-abb4-afa63274c0e6","owner":[],"postedDate":"July 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-30T14:09:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-28 08:06:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6726043","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6726043","identity":"rs-6726043","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}