Inflammation and blood-brain barrier breach remote from the primary injury following neurotrauma
Following injury to the central nervous system, increased microglia, secretion of pro- and anti-inflammatory cytokines, and altered blood-brain barrier permeability, a hallmark of degeneration, are observed at and immediately adjacent to the injury site. However, few studies investigate how regions remote from the primary injury could also suffer from inflammation and secondary degeneration.
Adult female Piebald-Viral-Glaxo (PVG) rats underwent partial transection of the right optic nerve, with normal, age-matched, unoperated animals as controls. Perfusion-fixed brains and right optic nerves were harvested for immunohistochemical assessment of inflammatory markers and blood-brain barrier integrity; fresh-frozen brains were used for multiplex cytokine analysis.
Immediately ventral to the optic nerve injury, immunointensity of both the pro-inflammatory biomarker inducible nitric oxide synthase (iNOS) and the anti-inflammatory biomarker arginase-1 (Arg1) increased at 7 days post-injury, with colocalization of iNOS and Arg1 immunoreactivity within individual cells. CD11b+ and CD45+ cells were increased 7 days post-injury, with altered BBB permeability still evident at this time. In the lower and middle optic tract and superior colliculus, IBA1+ resident microglia were first increased at 3 days; ED1+ and CD11b+ cells were first increased in the middle and upper tract and superior colliculus 7 days post-injury. Increased fibrinogen immunoreactivity indicative of altered BBB permeability was first observed in the contralateral upper tract at 3 days and middle tract at 7 days post-injury. Multiplex cytokine analysis of brain homogenates indicated significant increases in the pro-inflammatory cytokines, IL-2 and TNFα, and anti-inflammatory cytokine IL-10 1 day post-injury, decreasing to control levels at 3 days for TNFα and 7 days for IL-2. IL-10 was significantly elevated at 1 and 7 days post-injury with a dip at 3 days post-injury.
Partial injury to the optic nerve induces a complex remote inflammatory response, characterized by rapidly increased pro- and anti-inflammatory cytokines in brain homogenates, increased numbers of IBA1+ cells throughout the visual pathways, and increased CD11b+ and ED1+ inflammatory cells, particularly towards the synaptic terminals. BBB permeability can increase prior to inflammatory cell infiltration, dependent on the brain region.
KeywordsNeurotrauma Secondary degeneration Microglia Macrophages Cytokines Blood-brain barrier
Analysis of variance
Central nervous system
Degrees of freedom
Granulocyte-macrophage colony-stimulating factor
Inducible nitric oxide synthase
Optimal cutting temperature
Tumor necrosis factor alpha
Following an injury to a white matter tract in the central nervous system (CNS), axons located at the primary injury site are lesioned and degenerate . Neurons and glia spatially separated from the site of initial injury to axons undergo further damage and activation due to secondary degeneration . Inflammation following CNS trauma is responsible for both beneficial and detrimental effects, contributing to secondary damage but also facilitating neurorepair . Activation of resident microglia, secretion of immune mediators such as pro- and anti-inflammatory cytokines/chemokines, and infiltration of blood leukocytes characterize this robust inflammatory response . The release of reactive oxygen species (ROS) is a key pro-inflammatory feature, leading to glial activation and oxidative damage in the tissue surrounding the initial injury site  early after injury [6, 7]. Protective anti-inflammatory responses include myelin debris phagocytosis [8, 9] and neurotrophic and growth factor secretion . Astrocyte activation leads to a breach of the blood-brain barrier (BBB), with increased monocyte extravasation  and associated increases in Ca2+ and oxidative cascades of injury to both axons and glia, further contributing to spreading of degeneration .
Because macrophages and microglia are key modulator and effector cells in the immune response, much attention has been devoted to defining the dynamics of pro- and anti-inflammatory effects of trauma-induced inflammation within the linear constraints of microglia/macrophage polarization phenotypes, namely anti-inflammatory “M1” vs pro-inflammatory “M2” [5, 13, 14, 15, 16, 17]. The in vivo environment is complex and presents a variety of pro- and anti-inflammatory stimuli simultaneously [18, 19, 20]. In fact, recent in vivo studies of inflammation following CNS trauma have highlighted the plasticity of these cells where the same cell population is able to switch between polarization states in response to mediators in their surrounding microenvironment, and yet, others report the concurrent expression of both pro- and anti-inflammatory markers by a single cell [21, 22, 23, 24]. These findings suggest a lack of exclusivity in microglia/ macrophage activation states in vivo and indicate that the same cell responsible for promoting neuroinflammation can also release factors that promote wound healing. It is likely that the combination and balance of multiple factors (i.e., glial cells, immune cells, pro- and anti-inflammatory cytokines/ chemokine levels, time elapsed after injury, and lesion magnitude) will lead to different degrees of damage, as has been described in a range of injury models including glaucomatous damage at the optic nerve head, partial optic nerve transection, and spared nerve injury [25, 26, 27]. Furthermore, it is increasingly understood that specific monocyte, macrophage, and other glial and endothelial cell-derived cytokines play important roles in maintaining or perturbing BBB permeability, thereby further modulating the injury response . Characterizing the magnitude, timing, and duration of expression of these mediators has the potential to offer mechanistic insight and aid in the development of novel therapeutics.
Despite the plethora of research on trauma-induced inflammation at the site of a primary injury and immediately adjacent, relatively few studies investigate how regions remote from the primary injury site could also suffer from secondary degeneration and inflammation following neurotrauma [29, 30, 31]. Clinical studies suggest a link between remote damage and symptomatology, a phenomenon termed “diaschisis,” which contributes to functional impairments [32, 33, 34]. Previous studies in the spinal cord have shown that remote injury induces substantial changes in microglia and astrocytes, which contribute to central inflammatory responses [35, 36]. In addition, peripheral nerve injury evokes trafficking of immune cells from the circulation into the spinal cord parenchyma . Using our in vivo partial optic nerve transection model of secondary degeneration in adult Piebald-Viral-Glaxo (PVG) female hooded rats, we have previously demonstrated that a remote injury in the CNS (optic nerve) can cause a transient opening of the BBB in the visual pathways of the brain as early as 1 day following injury, with Evans blue fluorescence visible around the optic chiasm, optic tract, lateral geniculate nucleus, and superior colliculus. BBB permeability was maximal at 3 days following injury and decreased to levels not significantly different from uninjured controls at 7 days . An initial analysis of the inflammatory response early (1 day) following injury indicated that ED1+ and IBA1+ microglia/macrophage activation occurred along the length of the injured optic nerve, in the optic chiasm, and adjacent to the cerebral ventricles , and earlier studies have shown increases in these cells immediately adjacent to the injury at 3 days . However, a quantitative assessment of a range of inflammatory cell subtypes along the optic tract and into the superior colliculus is lacking. Furthermore, the spatiotemporal relationship between inflammation and blood-brain barrier breach remote to an injury is unknown. Specifically, it is not yet clear whether inflammatory cells that rapidly infiltrated the injury site spread along the optic nerve and tract into the brain, thereby initiating BBB breakdown. Alternative hypotheses include axonal degeneration and compromise of axonal transport initiating remote inflammation and BBB breakdown close to synaptic terminals in the superior colliculus or systemic changes contributing to a widespread inflammatory response. Here, we assess microglia and macrophage dynamics together with indicators of BBB breach and cytokine profiles, at various sites distal to a partial optic nerve transection injury, focusing on identifying the time at which changes are first observed in order to determine in a region-specific manner, whether blood-brain barrier breach precedes inflammation.
Adult female PVG hooded rats (160–180 g) were bred at the Animal Resources Centre (Murdoch, WA, Australia), housed under a standard 12-h light/dark cycle and fed standard rat chow and water ad libitum. Procedures adhered to the National Health and Medical Research Council Australian Code of Practice for the care and use of animals for scientific purposes and were approved by the University of Western Australia’s Animal Ethics Committee, approval numbers RA3/100/1201, RA3/100/673, and RA3/100/1485. The rats were anesthetized intraperitoneally with a combination of xylazine (Ilium Xylazil, Troy Laboratories, 10 mg/kg) and ketamine (Ketamil, Troy Laboratories, 50 mg/kg), and euthanized with an intraperitoneal injection of Euthal (pentobarbitone sodium 850 mg/kg; phenytoin sodium 125 mg/kg; Virbac).
The partial optic nerve transection procedure was similar to that described previously . Briefly, anesthetized rats were shaved along the skull towards the right eye. Under aseptic conditions, the skin was incised and retracted, and the right optic nerve accessed by deflecting the Harderian lachrymal gland immediately behind the right eye. The nerve parenchyma was exposed about 1 mm behind the eye by making a slit in the dura mater with ophthalmic scissors. A controlled 200-μm cut was made in the dorsum of the right optic nerve using a diamond radial keratotomy knife (Geuder), with the depth determined by the protrusion of the blade beyond a surrounding guard. Care was taken not to stretch the optic nerve or damage major ophthalmic blood vessels. The deflected tissue was replaced, and the skin was sutured with 4/0 non-absorbable silk suture. The eyes were lubricated with Luxyal eye drops, and the rats were placed on a warming blanket for recovery with subcutaneous injections of analgesic (Carprieve, 5 mg kg−1 in sterile water, Norbrook Australia, Pty. Ltd.; Victoria, Australia) and sterile PBS (1 ml). No postoperative infections were observed.
Summary of cohorts and numbers of animals per group
- Microglia/macrophages in the optic nerve
n = 10*
n = 10
n = 10
n = 10
- BBB analyses
n = 9–12 EB
n = 9–12 EB
n = 9–12 EB
n = 9–12 EB
n = 9–12 EB
- Microglia/macrophages in the optic tract
n = 9
n = 6 no EB
n = 6
n = 5–6
n = 4–5
n = 4
n = 8
n = 8
n = 8
n = 8
Immunohistochemistry, microscopy and analysis
Animals for BBB analyses (cohort 2) were administered a 2% (w/v) sterile solution of Evans blue (Sigma, E2129_10G) in PBS (2.8 ml/kg) via tail vein injection 1 h prior to euthanasia. Immediately following euthanasia, the animals for cohorts 1 and 2 (BBB and immunohistochemical assessments) were transcardially perfused-fixed with 0.9% (w/v) saline followed by 4% (w/v) paraformaldehyde (Sigma-Aldrich; St. Louis, Missouri, USA) in 0.1 M phosphate buffer, pH 7.2–7.4. The right optic nerves and brains were harvested and post-fixed in 4% paraformaldehyde for 1 h (nerves) or overnight (brains) and then cryoprotected in 15% sucrose in PBS and stored at 4 °C. The optic nerves were cryosectioned transversely (14 μm thickness), and the brains were cryosectioned coronally (20 μm thickness) and collected onto a series of slides so that each slide had a representative section from each area of interest. Sections for Evans blue analysis were collected onto Superfrost® Plus slides, air dried for an hour, and only briefly rinsed in PBS to reduce leakage of the dye from the sections before mounting using Fluoromount-G (Southern Biotech). The sections for immunohistochemistry were collected onto Superfrost® Plus slides and stored at − 80 °C.
Immunohistochemical analyses were conducted according to the previously described procedures . Primary antibodies used for immunohistochemical assessments of the optic nerves detected inducible nitric oxide synthase (iNOS) (1:400, BD Biosciences, ab3523), Arginase-1 (Arg-1) (1:400, Santa Cruz Biotechnology, sc-18355), CD45 (1:200, BD Pharmingen, 555482), and CD11b (1:400, 553310, BD Pharmingen). Primary antibodies used for immunohistochemical assessments of the brains (optic tracts and superior colliculus) recognized CD45 (1:500, Abcam, ab10558), CD11b (1:300, Sapphire Bioscience, ab2910), ionized calcium-binding adapter molecule 1 (IBA1) (1:1000, Abcam, ab5076), CD68 (ED1) (1:1000, Millipore Corporation, MAB1435), Caveolin-1 (1:400, Sapphire Bioscience, Ab2910), and Fibrinogen (1:500, HiTech Group Australia, A008002). Secondary antibodies were species-specific AlexaFluor 488-, 555-, and 647-conjugated antibodies (1:500, Invitrogen Australia). All tissue sections for a particular immunohistochemical outcome were processed at the same time.
Fluorescence imaging was performed using a Nikon Si1 inverted microscope (Nikon Corporation, Japan) for the right optic nerve and optic tract assessments of microglia and macrophages and a Leitz Diaplan microscope with an Olympus DP70 camera controlled by DP controller (version 220.127.116.11) for superior colliculus; a confocal Nikon C2 mounted on an upright Ni-E microscope, for assessments of BBB integrity (Evans blue fluorescence, fibrinogen, and caveolin-1 immunoreactivity), or for immunohistochemical assessments of microglia and macrophages in the brain (optic tracts and superior colliculus). Images from the Nikon microscopes were captured in a stack of 13 optical slices at 0.5 μm increments along the z-axis, obtained from the middle 6 μm of the section, controlled by NIS elements 4.3 software. All images for assessment of each outcome measure were captured at a constant exposure, using consistent microscope settings.
Cytokine measurements—multiplex assay
Rats from cohort 3 (n = 8 per group) were euthanized and transcardially perfused with ice-cold 0.9% saline, after which the brains were harvested, washed with ice-cold PBS, and homogenized in ice-cold RIPA buffer (Sigma Aldrich) supplemented with 1× protease inhibitor cocktail (Sigma Aldrich) (10 ml). Brain homogenates were incubated on ice for 20 min to aid protein solubilization, after which they were centrifuged at 12000g for 20 min at 4 °C, and the lysates were stored at − 80 °C until use. Total protein concentration of the brain lysates from each animal was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Pty Ltd.) according to the manufacturer’s instructions.
The concentration of ten cytokines (GM-CSF, Interleukin (IL)-12, Interferon (IFN)-γ, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α) in brain lysates was determined using the Cytokine Rat Magnetic 10-Plex Panel kit (Life Technologies) according to the manufacturer’s instructions using the Luminex® 200™ system (CMCA). Briefly, brain lysates were thawed, clarified by centrifugation (1000g for 10 min at 4 °C), and standardized at 500 μg/ml protein using assay diluent provided in the Rat Cytokine 10-Plex kit. Lyophilized cytokine standards (Rat Cytokine 10-Plex kit) were reconstituted in assay diluent (Rat Cytokine 10-Plex kit), and 1:3 serial dilutions were performed with assay diluent as instructed (Rat Cytokine 10-Plex kit) to give seven standard solutions. Colored magnetic beads conjugated with cytokine antibodies (Rat Cytokine 10-Plex kit) were reconstituted in 1× wash solution (Rat Cytokine 10-Plex kit) to give a 1× antibody bead solution, which was then pipetted into 80 wells of a 96-well plate (25 μl/well). The antibody beads were washed twice with 1× wash solution (200 μl/well). Following washing, prepared incubation buffer (Rat Cytokine 10-Plex kit) was added to each of the 80 wells (50 μl/well). Seven cytokine standard solutions were added in duplicate to wells designated for standards (100 ul/well), assay diluent was added in duplicate to wells designated for blanks (100 μl/well), and the 32 brain lysate samples from each animal (500 μg/ml) were added in duplicate to wells designated for samples (50 μl of sample + 50 μl of assay diluent), following which the plate was incubated at room temperature on a shaking platform (2 h) to enable cytokines to bind to their respective antibody beads. Following incubation, the wells were washed twice with 1× wash solution (200 μl/well), and subsequently, a 1× biotinylated antibody solution (Rat Cytokine 10-Plex kit) containing a mixture of biotinylated antibodies against each cytokine was added to each of the 80 wells (100 μl/well) and incubated at room temperature on a shaking platform (1 h). Following incubation, the wells were washed twice with 1× wash solution (200 μl/well), and subsequently, a 1× streptavidin-phycoerythrin solution (Rat Cytokine 10-Plex kit) was added to each of the 80 wells (100 μl/well) and incubated at room temperature on a shaking platform (30 min). Following incubation, the wells were washed thrice with 1× wash solution (200 μl/well) and stored in 1× wash solution (125 μl/well) overnight at 4 °C. The following day, working wash solution was decanted, and fresh 1× working wash solution was added (125 μl/well). The plate was then placed on a shaking platform (3 min) and inserted into an XY platform on the Luminex® 200™ system (CMCA) to measure cytokine concentrations for each sample calculated against the standard curve. Each well was sampled using a volume of 75 μl and a bead count of 100 per analyte before advancing to the next well.
Data analysis and statistics
Data are presented as mean ± standard error of mean. Statistical analyses of activated microglia and macrophage densities, Arg1 and iNOS immunointensities, and caveolin-1 and fibrinogen immunoreactivity were performed on SPSS (IBM) software, using one-way ANOVA or two-way ANOVAs to compare regions at a particular time point. For two-way ANOVAs, overall p, F, and df values are provided, with p, F, and df values provided for subsets where significance was reached. Tukey post hoc tests were used for optic nerve data and Sidak post hoc tests for optic tract data. If Levene’s test indicated inequality of variances, data were log or square root transformed for two-way ANOVAs or Games-Howell post hoc tests employed for one-way ANOVAs. Post hoc p values were provided where significance was reached using ANOVAs. Statistical analyses of cytokine data were performed on GraphPad Prism 6 software using one-way ANOVA to compare means of n = 8 animals at each time point, conducted individually for each cytokine, using Tukey post hoc tests.
Inflammatory responses in optic nerve immediately adjacent to the primary injury
Inflammatory cell responses in the brain
Blood-brain barrier integrity
Caveolin-1 is a structural component of caveolae, mediates transcytosis through endothelial cells via interactions with claudin , and can be used as an indicator of BBB integrity . Caveolin-1 immunoreactivity surrounding the third ventricle was assessed at 3, 7, 14, and 28 days following injury, to provide insight regarding the time of closure of the BBB following remote injury. The percentage of the third ventricle lined with caveolin-1+ cells was not different to control at 3, 7, and 28 days following partial optic nerve transection but was significantly reduced at 14 days (ANOVA: p = 5 × 10−4, F = 6.52, df = 4; 14 days p = 5 × 10−3) (Fig. 10e, f), indicating a transient compromise of caveolin-1-mediated junctions at this later time point.
Cytokine expression is altered in rat brains in a time-dependent manner following partial injury to the optic nerve
Delayed secondary damage often occurs to remote brain regions that are anatomically connected to the primary infarct by axonal projections. Studies in rats using middle cerebral artery occlusion as an ischemia model consistently show that distinct regions of the thalamus with axonal connections to the primary infarct develop gliosis, inflammation, and significant neuronal loss at delayed post-stroke time points . In our current study, a commonality among all observations of altered inflammatory cell density and BBB permeability in the brain is the axonal connectedness of these regions with the primary injury site, i.e., most significant alterations are observed in the contralateral optic tract and superior colliculus, which house the majority of injured axons that cross over at the optic chiasm, suggesting a role for axonal degeneration and potential white matter pathology, suggested to be of functional relevance for post-stroke recovery . Our findings indicate that remote damage is limited to areas connected to the primary injury site. However, it is important to note some limitations of the current study. Only one section per brain region was analyzed, and we did not examine all visual projections, such as the lateral geniculate nucleus. In addition, the analyses of cytokines used whole brain homogenates and thus were not confined to axonally connected pathways. Nevertheless, we observed a significant increase in the anti-inflammatory cytokine, IL-10, at 7 days post-injury, which coincided with increased numbers of microglia in the brain, perhaps indicating a concerted response by anti-inflammatory cell subtypes at this time. Depletion of CD11b+ cells worsens damage 24 h following ischemic injury, associated with increased pro-inflammatory indicators, and similar mechanisms may act here . Further, more focused assessments may provide additional insight. It is also important to note that PVG rats have particular susceptibilities to immune challenges . Indeed, there are strain-specific differences for many outcomes in rats . PVG rats were initially chosen for our studies using the partial optic nerve transection model of secondary degeneration, as they are not albino and therefore have better vision for assessments of visual function following injury. Female rats were chosen for ease of housing and to provide much needed and oft-missing information on injury outcomes in female animals.
Using Evans blue dye, we have observed breaches in the BBB in the brain as early as 1 day after this partial optic nerve injury , at the same time as both the current observed increases in both pro- (IL-2, TNFα) and anti- (IL-10) inflammatory cytokines in whole brain homogenates. While IL-2 and TNFα can cross the BBB [49, 50], and IL-10 cannot  the breached BBB may have facilitated entry of cytokines, elevated as a result of optic nerve injury, into the brain from the circulation. The delayed increases in inflammatory cell numbers observed in the middle contralateral optic tract at 7 days post-injury coincided with a significant increase in the area of fibrinogen-positive signal in this brain region, indicating associated alterations in BBB permeability. Increases in microglia- and macrophage-derived cytokines including TNFα correlate with immune cell infiltration and have been implicated in the modulation of endothelial cell function  and changes in BBB permeability . Reactive oxygen species generated by macrophages  also compromise BBB integrity . Thus, while BBB breaches in the upper tract precede, and may contribute to increases in inflammatory cells, multiple factors may be contributing to a feed forward system exacerbating the damage. While it is clear from our earlier published work that infiltration of microglia and macrophages also occurs at the partial optic nerve injury site and spreads in an efferent direction towards the brain along the nerve vulnerable to secondary degeneration [38, 39], it now becomes apparent that as time passes, inflammation may also move from degeneration in the upper tract and synaptic terminals back towards the injury. The anti-inflammatory cytokine IL-10 increases in brain homogenates in a biphasic pattern, being elevated at 1 day, returning to control levels at 3 days and elevated at 7 days after injury, likely as a compensatory response, associated with the later changes in microglia/ macrophage activation and phagocytosis. Further studies assessing cytokines in specific brain regions identified in this study as sites of inflammatory cell infiltration, pooling tissue from multiple animals, will be required to determine the direct relationships between cytokine changes, cellular infiltrations, and BBB breach. At later times after injury, there is a disruption to caveolin-1 immunoreactivity lining the ventricles, indicating that compromise of the BBB is not swiftly resolved and that injury to the optic nerve has persisting remote consequences in the brain.
While the increase in CD45+ cells in the optic nerve was transient, decreasing to control levels at 28 days following injury, the number of CD11b+ cells in the ventral optic nerve remained elevated at 28 days following injury. Similarly, iNOS and Arg1 expression were significantly increased within the ventral optic nerve 7 days after injury compared to uninjured controls, with only iNOS levels decreasing to control at 28 days post-injury. Interestingly, cells showed concurrent expression of iNOS and Arg1 indicating that these immunoreactive cells exhibit a mixed phenotype, as previously shown following traumatic brain injury . Similar findings have also been reported in the cortex following spared nerve injury to the sciatic nerve . As both microglia and infiltrating non-myeloid-derived leukocytes express CD11b and CD45 markers, but differ in the levels of expression of these markers, i.e., infiltrating leukocytes have high CD45 expression compared to resident microglia, we were unable to differentiate microglia from macrophages and leukocytes in general in this instance. Interestingly, CD45+ cells were not elevated in the optic tract, indicating differences in the features of the adjacent and remote inflammatory responses.
In summary, our study demonstrates that a partial injury to the optic nerve induces transient changes in whole brain cytokine expression and increased inflammatory cell density and BBB permeability remotely along the visual pathways in the brain. Significant alterations are restricted to the contralateral side of the brain, suggesting that, at least in this traumatic injury model, inflammatory and BBB damage is confined to regions that are “axonally connected” to the primary injury site, although it remains to be determined whether such damage also extends trans-synaptically.
We are grateful to Dr. Diwei Ho for generating the scientific illustrations.
National Health and Medical Research Council (NHMRC), Australia (APP1082403), and Neurotrauma Research Program, WA. MF is supported by an NHMRC Career Development Fellowship (APP1087114). The funding bodies played no part in the conduct of the study.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
NS, MG, AG, CB, and CAB conducted the experiments. NS, MG, AG, CB, TM, AB, TC, and MF analyzed the data. NS, MF, and MG wrote the manuscript. NS, MG, CAB, SI, SD, and MF designed the study, provided intellectual input, and edited the manuscript. All authors approved the final manuscript.
Ethics approval and consent to participate
Procedures adhered to the National Health and Medical Research Council Australian Code of Practice for the care and use of animals for scientific purposes and were approved by the University of Western Australia’s Animal Ethics Committee, approval numbers RA3/100/1201, RA3/100/673, and RA3/100/1485.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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