Acute neuroinflammation induces AIS structural plasticity in a NOX2-dependent manner
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Chronic microglia-mediated inflammation and oxidative stress are well-characterized underlying factors in neurodegenerative disease, whereby reactive inflammatory microglia enhance ROS production and impact neuronal integrity. Recently, it has been shown that during chronic inflammation, neuronal integrity is compromised through targeted disruption of the axon initial segment (AIS), the axonal domain critical for action potential initiation. AIS disruption was associated with contact by reactive inflammatory microglia which wrap around the AIS, increasing association with disease progression. While it is clear that chronic microglial inflammation and enhanced ROS production impact neuronal integrity, little is known about how acute microglial inflammation influences AIS stability. Here, we demonstrate that acute neuroinflammation induces AIS structural plasticity in a ROS-mediated and calpain-dependent manner.
C57BL/6J and NOX2−/− mice were given a single injection of lipopolysaccharide (LPS; 5 mg/kg) or vehicle (0.9% saline, 10 mL/kg) and analyzed at 6 h–2 weeks post-injection. Anti-inflammatory Didox (250 mg/kg) or vehicle (0.9% saline, 10 mL/kg) was administered beginning 24 h post-LPS injection and continued for 5 days; animals were analyzed 1 week post-injection. Microglial inflammation was assessed using immunohistochemistry (IHC) and RT-qPCR, and AIS integrity was quantitatively analyzed using ankyrinG immunolabeling. Data were statistically compared by one-way or two-way ANOVA where mean differences were significant as assessed using Tukey’s post hoc analysis.
LPS-induced neuroinflammation, characterized by enhanced microglial inflammation and increased expression of ROS-producing enzymes, altered AIS protein clustering. Importantly, inflammation-induced AIS changes were reversed following resolution of microglial inflammation. Modulation of the inflammatory response using anti-inflammatory Didox, even after significant AIS disruption occurred, increased the rate of AIS recovery. qPCR and IHC analysis revealed that expression of microglial NOX2, a ROS-producing enzyme, was significantly increased correlating with AIS disruption. Furthermore, ablation of NOX2 prevented inflammation-induced AIS plasticity, suggesting that ROS drive AIS structural plasticity.
In the presence of acute microglial inflammation, the AIS undergoes an adaptive change that is capable of spontaneous recovery. Moreover, recovery can be therapeutically accelerated. Together, these findings underscore the dynamic capabilities of this domain in the presence of a pathological insult and provide evidence that the AIS is a viable therapeutic target.
KeywordsAxon initial segment NOX2 Calpain Reactive oxygen species Neuroinflammation Microglia
Axon initial segment
Central nervous system
Experimental autoimmune encephalomyelitis
- Fizz-1 (Retnlb)
Hank’s balanced salt solution
Ionized calcium-binding adapter molecule 1
Mannose receptor, C type 1
Voltage-gated sodium channel 1.6
Neuronal nuclei, Rbfox-3
NADPH oxidase 2, gp91 phox
Phosphoglycerate kinase 1
Reactive oxygen species
Transforming growth factor beta
Tumor necrosis factor alpha
The axon initial segment (AIS) is a highly specialized axonal domain responsible for action potential initiation and modulation . The AIS is characterized by a unique assembly of cytoskeletal and scaffold proteins  and densely packed voltage-gated ion channels, which are recruited to and clustered at the AIS via the scaffolding protein ankyrinG (ankG) . ankG is considered the “master organizer” of the AIS and is essential for AIS function [4, 5]. Accumulating evidence suggests that the AIS is a dynamic domain capable of structural plasticity, undergoing changes in length , location [2, 7, 8], and ion channel clustering [9, 10] in response to neuronal pathology and altered activity.
AIS plasticity is characterized by the relocation of cytoskeletal-associated proteins such as ankG, βIV spectrin, neurofascin, and voltage-gated sodium (NaV) channels [2, 7, 11, 12, 13]. Although plasticity can be triggered by both pathologic and non-pathologic stimuli, the mechanisms and cell types that drive plasticity remain largely unknown. Schafer et al.  were the first to implicate the calcium-dependent protease calpain as a mediator of AIS plasticity with recent studies confirming these findings [10, 15]. Consistent with calpain activation, Evans et al.  reported that AIS plasticity is triggered by calcium channel activation with downstream activation of calcineurin. Recently, it has also been shown that microglia may influence neuronal activity through specific association with the AIS . Microglia-AIS contact was found to occur early in development and persist throughout adulthood in the uninjured brain  as well as during chronic inflammation present in an animal model of multiple sclerosis known as experimental autoimmune encephalomyelitis (EAE) , suggesting an important interaction that may influence AIS integrity.
Microglia, the resident immune cells of the central nervous system (CNS), are dynamic cells that survey, respond, and shape neuronal networks through neuronal contact and synaptic pruning [18, 19, 20, 21]. Microglia are critical for maintaining tissue homeostasis in the CNS, rapidly activating and eliminating pathogens and cellular debris in response to infection or insult [22, 23, 24]. Upon activation, microglia display an enhanced pro-inflammatory response and a dampened resolving phenotype [25, 26, 27]. This is typified by increased expression of inflammatory mediators such as tumor necrosis factor alpha (Tnf-α), cyclooxygenase-2 (COX-2), and NADPH oxidase 2 (NOX2), elevated production of reactive oxygen species (ROS), and reduced expression of resolving factors such as transforming growth factor beta (TGF-β), mannose receptor, C type 1 (Mrc1), and resistin-like beta (Fizz-1) [28, 29, 30]. Though reactive microglia play an important role in pathogen clearance and CNS homeostasis, amplified ROS production or aberrant activation of the inflammatory phenotype has been implicated in a number of neuronal pathologies [31, 32, 33, 34, 35] where AIS disruption is observed [14, 16, 17, 36, 37]. Previous studies from our lab demonstrated that chronic neuroinflammation in EAE resulted in changes in AIS length and protein clustering and this disruption corresponded with increased microglial reactivity and production of pro-inflammatory factors . Furthermore, AIS disruption corresponded with increased contact between reactive microglia and the AIS, suggesting that in a chronic inflammatory environment, pro-inflammatory microglia may drive AIS disruption .
The microglial inflammatory response is amplified by the enzyme NOX2, which is responsible for the microglial respiratory burst and extracellular production of ROS . NOX2 activity has been implicated in the chronic activation of microglia and its deleterious effects both through the production of extracellular ROS and through amplification of the pro-inflammatory response [39, 40, 41]. Inhibition of NOX2 reduced microglial ROS production and reduced microglia-mediated neurotoxicity [40, 42, 43]. Here, we investigate the role of microglial inflammation and the ROS-producing enzyme NOX2 on AIS integrity. Using a lipopolysaccharide (LPS)-induced model of neuroinflammation, we demonstrate that in the presence of acute microglial inflammation, AIS ankG clustering is disrupted and upon resolution of inflammation, AIS changes are reversed. Furthermore, ablation of NOX2 preserved AIS integrity. These data underscore the dynamic capabilities of the AIS in the presence of a pathological insult.
Six- to eight-week-old C57BL/6J mice and NOX2-deficient (B6.129S-Cybbtm1Din/J, NOX2−/−) mouse breeding pairs were purchased from Jackson Laboratories (Bar Harbor, ME). All animals were maintained in the AAALAC-accredited McGuire Veterans Affairs Medical Center (VAMC) vivarium with access to food and drink ad libitum. NOX2−/− mice have a targeted mutation of the 91-kD subunit of the oxidase cytochrome b and lack phagocyte superoxide production . NOX2−/− mice are maintained on a C57BL/6J background; therefore, age-matched C57BL/6J mice (NOX2+/+) were used as controls. All procedures were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the McGuire VAMC Institutional Animal Care and Use Committee.
Lipopolysaccharide (LPS; O111:B4, lot: 2728527) was purchased from Calbiochem (San Diego, CA). Female C57BL/6J and NOX2−/− mice (8–12 weeks) were given a single intraperitoneal (IP) injection of LPS (5 mg/kg, 10 mL/kg) or vehicle (0.9% saline). The LPS dose was based on the previously established neuroinflammation model [45, 46] where peripheral inflammation rapidly transfers to the brain, resulting in elevated microglial cytokine and ROS production . Saline- and LPS-treated mice were analyzed at 6 h, 24 h, 3 days, 1 week, and 2 weeks post-injection to assess the effects of microglial reactivity and AIS integrity throughout the course of neuroinflammation.
Didox (3,4-dihydroxybenzohydroxamic acid) was obtained from Molecules for Health, Inc. (Richmond, VA). Didox, a ribonucleotide reductase inhibitor and free radical scavenger, is a multifunctional compound that inhibits DNA replication, suppresses NF-κB activation, reduces oxidative injury, and attenuates microglia/macrophage production of inflammatory cytokines and ROS-producing enzymes [47, 48, 49]. Based on previous studies [17, 50], Didox (250 mg/kg solubilized in 0.9% saline) or vehicle (0.9% saline, 10 mL/kg) was administered intraperitoneally beginning at 24 h post-LPS injection and continued for 6 days. Animals were taken for analysis 1 week post-LPS injection.
Calpain inhibitor administration
Calpeptin was obtained from Calbiochem (San Diego, CA). Calpeptin is a cell-permeable inhibitor of calcium-activated proteases calpain-1 and calpain-2, which have been implicated in targeted cleavage of AIS proteins and alterations in AIS structure [9, 10, 14]. Based on previous studies [51, 52, 53], Calpeptin (50 μg/kg) or vehicle (0.1% dimethyl sulfoxide in saline, 10 mL/kg) was administered subcutaneously 30 min prior to injection of LPS (5 mg/kg, 10 mL/kg, IP) or vehicle (0.9% saline, 10 mL/kg, IP). On days 1 and 2 post-LPS injection, mice received a second and third dose of Calpeptin (calpain inhibitor), respectively. Vehicle-, LPS + vehicle-, or LPS + Calpeptin-treated mice were analyzed at 3 days post-LPS injection to assess the effects of calpain activity on AIS integrity.
Animals were deeply anesthetized using 0.016 mL/g body weight of a 2.5% solution of avertin (2,2,2 tribromoethanol, Sigma-Aldrich, St. Louis, MO) in 0.9% saline (Sigma-Aldrich, St. Louis, MO) and transcardially perfused with 4% paraformaldehyde (Ted Pella, Redding, CA). Following perfusion, brains were removed and immersed in 0.1 M PBS containing 30% sucrose for 48 h, frozen in OCT compound (Sakura, Netherlands), and serially sectioned into 40-μm-thick coronal sections stored at −80 °C and immunolabeled as previously described  using the following antibodies: mouse monoclonal anti-ankyrinG (ankG; NeuroMab, Davis, CA; N106/36; 1:500), rabbit polyclonal anti-Iba-1 (Wako Chemicals, Richmond, VA; 019-19741; 1:1,000), mouse monoclonal anti-NeuN (Millipore, Billerica, MA; MAB377; 1:1000), mouse monoclonal anti-gp91-phox (Santa Cruz, Dallas, TX; sc-130543; 1:500), mouse monoclonal anti-NaV1.6 (NaV1.6; NeuroMab, Davis, CA; K87A/10; 1:200). All secondary antibodies were obtained from Invitrogen Life Technologies (Grand Island, NY; Alexa™ Fluor) and used at a dilution of 1:500.
Imaging and analysis
Imaging was performed on a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY) housed in the VCU Department of Anatomy and Neurobiology Microscopy Facility. For AIS number analysis, images were collected as previously described . Briefly, confocal z-stacks spanning an optical thickness of 25 μm, using a pinhole of 1 Airy disc unit and Nyquist sampling (optical slice thickness, 0.48 μm), were collected from neocortical layer V for each of six sections (spanning 1.1 mm anterior to the bregma to 2.5 mm posterior to the bregma) per mouse resulting in 12 images per animal (n = 4–6 animals per treatment group). Images were then processed and analyzed using FIJI (NIH ImageJ software). Settings were optimized by comparing manual AIS tracings (previously described by ) and FIJI automated counts; no significant difference was found between methods (data not shown). Once established, settings remained constant throughout analysis. Thresholds of maximum intensity projections of ankG labeling were automatically set using the Otsu threshold method , and AISs were quantified using the “Analyze Particles” plugin (FIJI) (size 0–infinity μm2; circularity 0–0.5; objects touching edges excluded). ankG-positive structures measuring <10 μm were excluded from analysis consistent with previous studies [17, 36, 37].
For analysis of microglial NOX2 immunoreactivity, confocal z-stacks spanning an optical thickness of 25 μm were collected from neocortical layer V for each of six sections (spanning 1.1 mm anterior to the bregma to 2.5 mm posterior to the bregma) per mouse (n = 3 animals per treatment group). Images were blinded, and NOX2 immunoreactivity in Iba-1+ cells was quantified using Volocity™ 3D Image Analysis Software version 6.3 allowing 3D confirmation of double immunolabeling in each Iba-1+ cell. The total number of microglia and the number of NOX2+ microglia were counted manually for each double-immunolabeled z-stack. Data are presented as the percent of NOX2+ microglia (Iba-1+) per field of view.
Neuronal density and cortical volume measurements from saline or LPS-injected mice
Average NeuN count
(% saline ± SEM)
Average cortical volume ± SEM
(μm3) × 103
100 ± 3.2
100 ± 7.3
1.4 ± 0.2
1.14 ± 0.3
LPS 6 h
103 ± 5.9
1.1 ± 0.2
LPS 24 h
98 ± 0.8
109 ± 4.9
1.1 ± 0.1
1.15 ± 0.3
LPS 3 days
108.1 ± 2.5
1.4 ± 0.1
LPS 1 week
97.7 ± 2.5
110 ± 3.4
1.2 ± 0.1
1.27 ± 0.1
LPS 2 weeks
108 ± 3.9
1.5 ± 0.1
Cortical volume analysis was performed using the Cavalieri principle as previously described (modified [55, 56]). Briefly, unbiased stereology was performed using every 15th section from the total sections spanning the cortical region 1.1 mm anterior to the bregma to 2.5 mm posterior to the bregma and analyzed to estimate cortical volume. Each reference space was outlined with a ×2 objective and analyzed using a point-grid analysis, sampling 100% of the regions of interest. Samples were counted in a blind manner and volumes calculated using an Olympus BX51 microscope (Center Valley, PA) and newCAST software (Visiopharm, Hoersholm, Denmark) (n = 3–4 mice per treatment group). No differences in cortical volumes were detected among any treatment groups (NOX2+/+ Saline, NOX2+/+ LPS-injected, NOX2−/− Saline, or NOX2−/− LPS-injected; Table 1).
Adult cortical microglia were isolated using MACS magnetic bead separation (Miltenyi Biotec, San Diego, CA) as described previously [17, 45]. Briefly, saline-treated and LPS-treated mice were deeply anesthetized and transcardially perfused with 50 mL ice-cold PBS. After removal of the meninges, the cerebral cortices of two mice were harvested and pooled per sample (2 mice = 1 n) and suspended in Hank’s balanced salt solution (HBSS) without CaCl2 and MgCl2 (Corning, Corning, NY). A single-cell suspension was prepared using the Miltenyi Neural Tissue Dissociation Kit according to the manufacturer’s instructions. The cells were depleted of myelin by suspension in 3 mL of 30% isotonic Percoll™ (GE Healthcare Life Sciences, Pittsburgh, PA) followed by a 10-min centrifugation at 700 x g at 4 °C. The cell pellet was washed in 5 mL HBSS without CaCl2 and MgCL2, and isolation of microglia was performed with magnetic CD11b microbeads (Miltenyi) and MACS magnetic separator (Miltenyi) according to the manufacturer’s instructions.
RNA isolation and RT-qPCR analysis
Oligonucleotide primer sets used for RT-qPCR
Calpain activity assay
To quantify the levels of calpain activity and to determine the effect of Calpeptin on inhibition of calpain activity, vehicle-, LPS + vehicle-, or LPS + Calpeptin-treated mice were deeply anesthetized and transcardially perfused with 50 mL ice-cold 0.9% saline at 3 days post-LPS injection. Cerebral cortices (10 mg) were harvested and immediately homogenized in ice-cold extraction buffer (Calpain activity kit). Samples were centrifuged for 5 min at 4 °C at 15,000 x g to remove insoluble material. Calpain activity was quantified using a fluorometric calpain activity assay kit (ab65308, Abcam, Cambridge, MA) according to the manufacturer’s protocol. All samples were analyzed in triplicate, and calpain activity was measured using a Tecan M1000 PRO microplate reader (Männedorf, Switzerland). Changes in calpain activity were normalized to saline control levels and expressed as relative fluorescent units (RFU).
All graphing and statistical analyses were performed using GraphPad Prism version 6.03 (GraphPad Software, San Diego, CA). Data were analyzed by a one-way or two-way analysis of variance and, where mean differences were significant, assessed using Tukey’s honest significance difference post hoc analysis. Treatment groups were presented as percent of saline control (% Control ± SEM), and p < 0.05 was considered statistically significant.
LPS-induced inflammation alters AIS protein clustering
AIS disruption is reversible
The AIS is the site of action potential initiation and thus is critical for neuronal function . Studies have shown that the AIS can undergo structural plasticity in development and in response to pathological insults to sustain proper signaling within neuronal networks [2, 6, 61, 62]. To determine in vivo if inflammation-induced AIS disruptions are reversible, we assessed ankG clustering of AISs in saline- and LPS-treated mice 2 weeks post-injection (Fig. 1). ankG immunolabeling revealed that the number of AISs in LPS-treated mice 2 weeks post-injection returned to baseline and was not significantly reduced compared to saline controls (91.3% ± 2.8) (Fig. 1f, g). Furthermore, AISs at 2 weeks post-LPS injection were significantly increased compared to LPS 1 week treated mice (mean difference 25.9% ± 5.7, p < 0.01) (Fig. 1e–g). Thus, LPS-induced disruption of AIS ankG clustering is reversible.
AIS integrity coincides with microglial inflammatory response
Treatment with anti-inflammatory Didox reverses AIS disruption
Treatment with Didox alters microglial NOX2
Ablation of NOX2-derived ROS production prevents AIS disruption
Inhibition of calpain prevents AIS disruption
In this study, we demonstrate that LPS-induced neuroinflammation disrupts protein clustering at the AIS concomitant with the microglial inflammatory response resulting in an ~30% loss of AIS detection. Importantly, we found that inflammation-induced AIS disruptions were reversed following resolution of microglial inflammation and changes in AIS ankG clustering are NOX2-mediated and dependent on calpain activity. Thus, in the presence of acute microglial inflammation, the AIS undergoes an adaptive change that is capable of spontaneous recovery, underscoring the dynamic capabilities of this domain in the presence of a pathological insult.
The AIS has the capacity to adapt and recover
The AIS is targeted for disruption in injury and disease emphasizing its need for homeostatic adaptations. Indeed, many studies [16, 17, 37, 71, 72, 73] have shown that the AIS is plastic, undergoing change in response to various stimuli. However, few studies have demonstrated that these changes are reversible. Alterations in AIS length  and location  caused by changes in neural activity were reversible in vitro; however, loss of AIS protein clustering due to ischemic insults in vitro were not, even in the absence of cell death . A previous study examining AIS integrity after stroke observed axonal sprouting resulting in an increase in small, immature AISs demonstrating reparative potential of this domain . Furthermore, our lab previously reported that shortening of AIS length is reversible following treatment with the anti-inflammatory Didox . Here, we provide evidence that loss of AIS protein clustering is spontaneously reversible, independent of axonal sprouting. Moreover, we show that by modulating the neuroinflammatory response using therapeutic intervention, the rate of AIS recovery can be increased, even after significant AIS disruption has occurred. These data suggest that while insults at the AIS, such as ischemia , can cause irreversible damage, the AIS has the capacity to adapt and recover after insult. The mechanism by which this occurs or what the extent of injury is after which the AIS cannot recover remains to be determined.
Microglial phenotype influences AIS integrity
Although AIS plasticity can be triggered by both pathological and non-pathological stimuli, the events that drive plasticity remain largely unknown. Recently, Baalman et al.  established a relationship between microglia and the AIS, revealing that microglia contact AISs early in development and throughout adulthood in the uninjured brain, suggesting an important interaction that may influence neuronal excitability. In a model of chronic neuroinflammation, reactive microglia increased contact with AISs, and this contact both preceded AIS disruption and increased with disease progression, suggesting that in a chronic inflammatory environment, increased microglial contact may drive AIS disruptions . Consistent with previous findings, we found that reactive microglia contact the AISs during LPS-induced neuroinflammation. However, contrary to findings in the chronic inflammatory model, the amount of contact made by microglia did not increase throughout the course of inflammation and did not correlate with AIS disruption (data not shown). The microglial inflammatory profile, however, did correspond with AIS disruption and recovery. Furthermore, modulation of the inflammatory profile using anti-inflammatory treatment increased the rate of AIS recovery. Though our findings suggest that changes in microglial inflammation correspond with AIS alterations, it is possible that these changes do not directly influence AIS integrity. However, the direct association of microglia with the AIS suggests this axonal domain may be particularly vulnerable to changes in microglial reactivity. Thus, our findings suggest that AIS integrity may be influenced by microglial phenotype, with a pro-inflammatory phenotype driving AIS disruption while a resolving phenotype hastens repair.
Consistent with this premise, Klapal et al.  showed that incubation of hippocampal cultures with activated microglia or the pro-inflammatory cytokine Tnf-α increased neuronal excitability. In contrast, incubation with the pro-resolution factor TGF-β decreased Na+ current density to control levels. Together, these findings suggest that neuroactive factors released by microglia augment neuronal excitability, which drives AIS structural changes [2, 6, 12]. Here, we demonstrate that AIS structure is altered following significant increases in microglial expression of Tnf-α. Furthermore, this AIS pathology is reversed after expression of microglial TGF-β is enhanced. Thus, our findings are consistent with microglial neuroactive factors driving changes in neuronal activity and AIS structural plasticity.
NOX2-mediated ROS, calpain, and AIS changes
During insult, pro-inflammatory microglia increase expression of inflammatory mediators and ROS-producing enzymes [30, 40, 45, 75]. ROS are highly reactive and diffuse signaling molecules that regulate cell functions through redox modification of target proteins. ROS can result in further production of reactive species [76, 77] and elevated calcium levels , which have been implicated in AIS disruption. In this study, we show that changes in microglial expression of ROS-producing enzymes correspond with AIS disruption and recovery, suggesting a role for microglial ROS in inflammation-driven AIS disruption. Consistent with this premise, ablation of NOX2 prevented AIS disruption. Though NOX2 is primarily expressed by microglia, NOX2 is also present in cortical neurons, where it plays a role in ROS regulation and calcium dynamics . Therefore, NOX2 ablation may preserve AIS integrity through both the prevention of microglial ROS release and neuronal NOX2 ROS production, both of which may converge on pathways resulting in AIS changes.
Reactive species such as hydrogen peroxide and nitric oxide influence calcium-permeable channels including L-type Ca(2+) [78, 80, 81] and TRPM channels [82, 83]. Upon activation, intracellular calcium concentrations rise, resulting in the subsequent activation of calcium-regulated proteins such as calpain [9, 10, 14], CamKII , and calcineurin [8, 12], which have been implicated in AIS disruption. Consistent with previous studies [9, 10, 14], our data implicate the calcium-dependent protease calpain as a mediator of AIS structural changes. In this study, we demonstrate that acute neuroinflammation increases calpain activity consistent with disruption of AIS ankG clustering and inhibition of calpain prevents inflammation-induced disruptions. Together, our data suggest that NOX2-derived ROS and calpain activity are drivers of AIS structural plasticity during acute neuroinflammation.
In conclusion, we demonstrate that in the presence of acute neuroinflammation, protein clustering at the AIS is altered. Importantly, our data demonstrate that this AIS disruption is reversible and that the AIS has the capacity to adapt and spontaneously recover. Furthermore, we reveal that inflammation-driven plasticity at the AIS is mediated by NOX2 and calpain activity.
The authors wish to thank Dr. Scott Henderson, Director of the VCU Department of Anatomy and Neurobiology Microscopy Facility, for his assistance with image collection and data analysis. The authors also wish to thank Dr. Howard Elford for his generous gift of the Didox reagent.
This study was supported by the Veterans Affairs Merit Grant (5IO1BX002565—JLD and 101BX001759—GHDV). The VA provided salary support for JL Dupree and GH DeVries. Microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported, in part, with funding from NIH-NINDS Center core grant 5P30NS047463.
Availability of data and materials
The datasets during and/or analyzed during the current study will be made available from the corresponding author on reasonable request.
SB was responsible for the experimental design, provided the required animal husbandry, conducted all the LPS injections, tissue preparation and analysis, and data interpretation, and was instrumental in the manuscript preparation. NG assisted in the development of automated AIS quantitation method, data collection, and analysis. BS assisted SB with the animal husbandry and tissue preparation. GD provided oversight for the study including experimental design, data interpretation, and manuscript preparation. JD provided oversight for all aspects of the study including experimental design, data interpretation, and manuscript preparation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All procedures were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the McGuire VAMC Institutional Animal Care and Use Committee.
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