S100B Inhibition Reduces Behavioral and Pathologic Changes in Experimental Traumatic Brain Injury

Abstract

Neuroinflammation following traumatic brain injury (TBI) is increasingly recognized to contribute to chronic tissue loss and neurologic dysfunction. Circulating levels of S100B increase after TBI and have been used as a biomarker. S100B is produced by activated astrocytes and can promote microglial activation; signaling by S100B through interaction with the multiligand advanced glycation end product-specific receptor (AGER) has been implicated in brain injury and microglial activation during chronic neurodegeneration. We examined the effects of S100B inhibition in a controlled cortical impact model, using S100B knockout mice or administration of neutralizing S100B antibody. Both interventions significantly reduced TBI-induced lesion volume, improved retention memory function, and attenuated microglial activation. The neutralizing antibody also significantly reduced sensorimotor deficits and improved neuronal survival in the cortex. However, S100B did not alter microglial activation in BV2 cells or primary microglial cultures stimulated by lipopolysaccharide or interferon gamma. Further, proximity ligation assays did not support direct interaction in the brain between S100B and AGER following TBI. Future studies are needed to elucidate specific pathways underlying S100B-mediated neuroinflammatory actions after TBI. Our results strongly implicate S100B in TBI-induced neuroinflammation, cell loss, and neurologic dysfunction, thereby indicating that it is a potential therapeutic target for TBI.

INTRODUCTION

Traumatic brain injury (TBI) represents a major public health problem, with >1.7 million new cases annually reported in the United States alone, according to the Centers for Disease Control and Prevention. TBI causes cell death and neurologic dysfunction through both physical disruption of the tissue (primary injury), as well as delayed molecular and cellular mechanisms that cause progressive white and gray matter damage (secondary injury) and may potentially continue for months to years. ¹ Such secondary injury mechanisms, such as chronic microglial and astrocyte activation, contribute to pathogenesis of brain injury, exacerbating neurologic dysfunction. ² Over the years, we have demonstrated the progressive and sustained nature of neuroinflammation through microglial and astrocyte activation, particularly in the cortex, using multiple experimental models of TBI.^3,4

One of the biochemical responses following glial activation induced by brain injury is increased circulating serum and cerebro-spinal fluid levels of the calcium-binding protein S100B, which is primarily expressed in the astrocytes and Schwann cells. ⁵ The mechanisms of action of S100B as an intracellular regulator are different from those as an extracellular factor. As an intracellular regulator, S100B stimulates cell proliferation and migration, inhibits apoptosis and differentiation, and activation of astrocytes, which may have implications for brain repair after central nervous system (CNS) injury. ⁶ In contrast, as an extracellular factor S100B interacts with advanced glycation end product-specific receptor (AGER) to exert beneficial or detrimental effects, or pro-proliferative or pro-differentiative consequences based on its concentration and microenvironment.^7,8 In addition to its expression in astroglia and Schwann cells, S100B has also been found in adipocytes, chondrocytes, cardiomyocytes, lymphocytes, bone marrow cells, and melanocytes where it exerts intracellular and extracellular functions. ⁶ Therefore, both CNS and circulatory S100B exert the same functions that are dependent on their concentration and may serve as important biomarkers of CNS injury. Intracellular S100B interaction with Src kinase activates phosphoinositide 3-kinase signaling and downstream RhoA/Rack-dependent stress fiber formation as well as glycogen synthase kinase 3β/Rho-dependent stellation. ⁹ In addition, S100B is also expressed in oligodendrocytes and cultured microglia/microglial cell lines, ¹⁰ but S100B-regulated intracellular events in these cells have not been elucidated. More importantly, the concentration of S100B and the state of the cell are two major variables that determine whether extracellular S100B exerts trophic or toxic effects. The overproduction of S100B by activated astrocytes after brain injury increases microglial and astrocyte activation, as well as neuronal cell death. ⁹ The release of S100B can stimulate the generation of oxidative stress-related enzymes and pro-inflammatory entities, such as inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 β (IL-1β), and IL-6. ⁹ Therefore, S100B is regarded as a biomarker of activation of pathologic mechanisms after brain injury and neuroinflammatory/neurodegenerative disorders.^11,12 The elevated levels of S100B in neurodegenerative models have been found to be associated with impairments in learning and memory function.^13,14 In addition, recently an association between elevated circulating S100B levels and impaired neurologic outcomes has been observed after clinical brain injury.^5,15

Studies suggest that cellular signaling resulting from the interaction of S100B with AGER may be responsible for its pro-inflammatory effects. ¹⁶ AGER is a member of the immunoglobulin (Ig) family of cell surface molecules that recognizes multiple ligands, including AGE, amphoterin, amyloid-β-peptide and β-fibrils, and S100B. Extracellular S100B has been observed to exhibit trophic effects on neurons by interaction with AGER,^7,8 which has been implicated in neuroprotection and neurodegeneration as well as in inflammatory responses. ¹⁷ S100B at high concentrations via acute stimulation of AGER causes neuronal apoptosis by excessive extracellular signal-regulated kinase 1 (ERK1)/2 activation and overproduction of reactive oxygen species (ROS). ⁷

In our study, we evaluated the effects of S100B inhibition using either a knockout model or administration of a neutralizing S100B antibody in a mouse model of controlled cortical impact (CCI). Furthermore, we investigated the potential interaction of S100B with AGER in CCI, as well as the effects of S100B on primary microglial cultures or a mouse microglial cell line after activation with lipopolysaccharide (LPS) or interferon gamma (IFNγ). Our study highlights the importance of S100B not only as a biomarker for TBI-induced injury but also as a potential therapeutic target.

MATERIALS AND METHODS

Controlled Cortical Impact

Our custom-designed CCI-injury device^3,18 consists of a microprocessor-controlled pneumatic impactor with a 3.5-mm diameter tip. Male mice were anesthetized with isoflurane (4% induction, 2% maintenance) evaporated in a gas mixture containing 70% N₂O and 30% O₂ and administered through a nose mask. The mouse was placed on a heated pad and a core body temperature was maintained at 37 °. A 10-mm midline incision was made over the skull, the skin and fascia were reflected, and a 4-mm craniotomy was made on the central aspect of the left parietal bone. Moderate injury was induced using an impactor velocity of 6 m/s and deformation depth of 2 mm, as previously detailed.^3,18 Sham animals underwent the same procedure as injured mice except for the impact.

Animals and treatment. All experiments involving animals were approved by the Institutional Animal Care and Use Committee at the University of Maryland and were performed following guidelines and regulations that ensured the humane care and use of animals in research. The manuscript was prepared in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines—all sections of the manuscript: from the study design, housing, sample size, and statistical analysis to the outcomes and interpretation were written as per ARRIVE guidelines. All mice were divided into different groups using a randomization protocol and by involving a blinded co-investigator to genotype/treatment to avoid any bias. Furthermore, assessment of all functional outcomes and analysis of stereological data was performed by an investigator blinded to the groups.

Genetic intervention: The S100B^−/− line (CD1 background) was a generous gift of Dr Alexander Marks (University of Toronto). ¹⁹ Genotyping was performed as previously described, ²⁰ and all experiments used male S100B^−/− and S100B^+/+ littermates (20 to 25 g, 3 months old).

Neutralizing anti-S100B antibody treatment: Male C57Bl/6 mice (20 to 25 g, 3 months old) were randomized into six groups and administered 10 mg/kg anti-S100B (BL356, Bethyl Laboratories, Montgomery, TX, USA) or an isotype matched IgG (Catalog no. P50-1000, Bethyl Laboratories) antibody at 24 hours and 14 days after injury, via the intraperitoneal route, based on preliminary pharmacokinetic data.

Beam walk. Chronic motor recovery following TBI was assessed using a beam walk test as previously described.^3,18 Mice were placed on one end of the beam and the number of foot faults of the right hindlimb was recorded over 50 steps. Mice were trained on the beam walk for 3 days before TBI and tested at 1, 7, 14, 21, and 28 days after injury.

Morris water maze (MWM). Spatial learning and working memory following TBI was assessed using the acquisition paradigm of MWM test on postinjury days (PIDs) 14, 15, 16, and 17, as described previously.^3,18 Spatial learning and memory performance was assessed by determining the latency (seconds) to locate the submerged hidden platform with a 90 second limit per trial. Reference spatial memory was assessed by a probe trial, with a 60-second limit, on PID 18. The time spent in the target quadrant was recorded.

Novel object recognition (NOR). Retention or intact memory was assessed by the NOR test on PID 21. The apparatus consists of an open field (22.5 × 22.5 cm²) with two adjacently located imaginary circular zones, as previously designed. ¹⁸ The zones were equally spaced from the sides in the center of the square and designated as “old object” and “novel object” zones using the AnyMaze video tracking system. On PID 20, all animals were placed in the open field for 5 minutes each without any objects present for habituation. Two 5-minute trials were performed on PID 21: the first (training) trial with two old objects in both zones and the second (testing) phase with one old object and one novel object present in the respective zones of the open field. There was an intertrial interval of 60 minutes. Retention memory was determined as the “Discrimination Index” (D.I.) for the second trial that was calculated using the following formula:

$\%\mathrm{D}.\mathrm{I}.=\frac{\text{Time}spentinnovelobjectzone}{(\text{Time}spentinoldobjectzone+Timespentinnovelobjectzone)}\times 100$

(1)

Lesion volume. Sections were stained with cresyl violet (FD Neuro-Technologies, Baltimore, MD, USA), dehydrated, and mounted for analysis at 28 days after TBI. Lesion volume was quantified based on the Cavalieri method of unbiased stereology using the Stereoinvestigator software (MBF Biosciences, Williston, VT, USA). ¹⁸ The lesion volume was quantified by outlining the missing tissue on the injured hemisphere using the Cavalieri estimator with a grid spacing of 0.1 mm. Out of the total 96 60-μm sections, every eighth section was analyzed beginning from a random start point.

Assessment of neuronal cell loss. The total number of surviving neurons was quantified in the cortex and thalamus using the optical fractionator method of unbiased stereology at 28 days after TBI, as described previously. ¹⁸ The optical dissector had a size of 50 × 50 μm² in the x and y axis, respectively, with a height of 10μm and guard zone of 4μm from the top of the section. A grid spacing of 400 μm in the x axis and 400 μm in the y axis was used, resulting in an area fraction of one-sixty-fourth. To assess neuronal cell loss in the cornu ammonis 1 (CA1), CA2, CA3, and dentate gyrus (DG) subregions of the hippocampus every fourth 60-μm section between − 1.22 and − 2.54 mm from bregma was analyzed beginning from a random start point. The optical dissector had a size of 50 × 50 μm² in the x and y axis, respectively, with a height of 10 μmand guard-zone of 4 μm from the top of the section. For the CA1, CA2, and CA3 subregions, a grid spacing of 75 μm in the x axis and 100 μm in the y axis was used, resulting in an area fraction of one-twelfth. For the DG subregion, a grid spacing of 175 μm in the x axis and 100 μm in the y axis was used, resulting in an area fraction of one-twenty-eighth. The volume of the hippocampal subfield was measured using the Cavalieri estimator method with a grid spacing of 50 μm. The estimated number of surviving neurons in each field was divided by the volume of the region of interest to obtain the neuronal cellular density, expressed as counts/mm³.

Assessment of microglial activation. Sections were stained with Iba-1 (Wako Chemicals, Richmond, VA, USA) and the activation status of the Iba-1-positive-stained microglial cells was analyzed based on morphologic features, as previously detailed. ³ Stereoinvestigator software was used to count cortical microglia in each of the three microglial morphologic phenotypes (surveillant—ramified, and activated—hypertrophic and bushy) using the optical fractionator method at 7 and 28 days after TBI. ²¹ The sampled region was the ipsilateral cortex between − 1.22 mm and − 2.54 mm from bregma and dorsal to a depth of 2.0 mm from surface. Every fourth 60-μm section was analyzed beginning from a random start point. The optical dissector had a size of 50 × 50 μm² in the x and y axis, respectively, with a height of 10μm and guard zone of 4μm from the top of the section. A grid spacing of 150μm in the x axis and 150μm in the y axis was used, resulting in an area fraction of one-ninth. The volume of the region of interest was measured using the Cavalieri estimator method with a grid spacing of 100μm for the cortex. The estimated number of microglia in each morphologic class was divided by the volume of the region of interest to obtain the cellular density expressed in counts/mm³.

Proximity ligation assays. Formalin-fixed, paraffin-embedded sections on glass slides collected from 7-day samples from TBI and sham groups were deparaffinized, rehydrated, and processed for proximity ligation assay using a DUOLink II Brightfield Kit (no. 92012, OLINK, Uppsala, Sweden) as previously described. ²² Primary antibodies included mouse anti-S100B (1:50 dilution of no. 612376, BD Transduction Laboratories, San Jose, CA, USA) and rabbit anti-AGER (1:20 dilution of no. AP4861a, Abgent, San Diego, CA, USA).

Microglia Cultures

Primary cortical microglia were obtained from postnatal day 2 mouse pups and cultured as described before. ²³ The BV2 murine microglia were grown and maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) at 37 ° in a humidified incubator under 5% CO₂. S100B (1 μmol/L; Sigma-Aldrich, St Louis, MO, USA) was added to microglia alone and for 1 hour before LPS (Sigma-Aldrich, indicated concentrations in μg/mL) or recombinant mouse IFNγ (R&D Systems, Minneapolis, MN, USA, indicated concentrations in ng/mL) stimulation.

Nitric oxide (NO) assay. NO production was assayed using the Griess Reagent Assay (Invitrogen, Grand Island, NY, USA), according to the manufacturer's instructions. Data were presented as the percentage of control-treated values.

Measurement of intracellular ROS. Intracellular ROS levels were measured by 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA). Briefly, microglia were incubated with 10 μm H₂DCFDA (Molecular Probes, Eugene, OR, USA) for 45 min at 37 ° in 5% CO₂. Fluorescence was measured using excitation and emission wavelengths of 490 and 535 nm, respectively. Data are presented as the percentage of control-treated values.

Statistical Analysis

The number of animals per group for each assessment was based on our prior studies using the CCI model^3,18,21 and satisfied power requirements. Quantitative data were expressed as mean ± s.e.m., and in vitro data were expressed as mean±s.d. Functional data for beam walk and MWM acquisition task, respectively, were analyzed by repeated-measures two-way analysis of variance (ANOVA) to determine interactions between PIDs, injury/sham groups, and effect of pharmacological or genetic intervention, followed by Tukey's post-hoc test. The data for MWM probe trial were analyzed by repeated-measures two-way ANOVA, and NOR task results were analyzed by one-way ANOVA followed by multiple pairwise comparisons using Tukey's post-hoc test. The lesion volume data were analyzed by unpaired Student's t test (genetic knockout study) or one-way ANOVA (anti-S100B IgG study). The stereological assessments of neuronal cell loss and microglial activation were analyzed by one-way ANOVA followed by Tukey's post-hoc test. In vitro studies were analyzed by one-way ANOVA followed by Tukey's post-hoc test. Data were analyzed using SigmaPlot 12 (Systat Software, San Jose, CA, USA) or GraphPad Prism Version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). A P < 0.05 was considered statistically significant.

RESULTS

Genetic Ablation of S100B Provides Neuroprotection

Behavioral outcomes. Functional assessment of fine motor coordination was performed at various time points after injury using a beam walk test (n = 11 to 14/group). TBI induced sensorimotor impairments at all time points when compared with sham-injured mice (Figure 1A;P < 0.001, vs. sham). However, S100B^−/− mice failed to show any improvement in sensorimotor function.

Figure 1.

S100B^−/− mice had improved retention memory function after traumatic brain injury (TBI). (A) TBI induced sensorimotor impairments at all time points when compared with sham-injured mice in the beam walk test (***P < 0.001, **P < 0.01, vs. sham). However, S100B^−/− mice failed to show any improvement in sensorimotor function. Analysis by repeated measures two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. (B) S100B^−/− mice showed significant improvement (⁺P < 0.05, vs. S100B+^/+) in Discrimination Index in the novel object recognition task. Analysis by one-way ANOVA followed by Tukey's post-hoc test. (Mean ± s.e.m.; n = 11 to 14/group).

Spatial learning and reference memory were assessed using the acquisition phase and probe trial of the MWM test, respectively.

TBI resulted in spatial learning impairments on PIDs 16 and 17 (P < 0.05, vs. sham, data now shown), with no statistically significant differences between the two sham groups. S100B^−/− mice did not show improvements in cognitive performance in the acquisition phase as well as probe trial of the MWM test (data not shown). Retention or intact memory function was evaluated using the NOR task. The data were expressed as the D.I. and statistically analyzed by one-way ANOVA followed by Tukey's post-hoc test ((F (3,32) = 7.308;P = 0.00007)). S100B^+/+ mice showed significant impairments in retention memory function (Figure 1B;P < 0.05, vs. sham). In contrast, S100B^−/− mice showed a significant improvement in retention memory function when compared with S100B^+/+ mice (Figure 1B;P < 0.05, vs. S100B^+/+).

Neuropathologic changes. TBI-induced lesion volume (n = 4 to 7/group) was estimated by unbiased stereological techniques. Histologic assessment showed that S100B^+/+ mice developed a large lesion following TBI (Figures 2A and 2B, 6.06±0.62 mm³), whereas S100B^−/− mice showed a significant reduction in lesion size (P < 0.05, vs. S100B^+/+, 3.56 ±0.53 mm³).

Figure 2.

S100B^−/− mice had reduced lesion volume but no reduction in neuronal cell loss after traumatic brain injury (TBI). (A) Histologic assessment using unbiased stereology showed that S100B^+/+ mice developed a large lesion following TBI, whereas S100B^−/− mice showed a significant reduction in lesion size (*P < 0.05, vs. S100B^+/+). Analysis by one-tailed unpaired Student's t-test. (B) Representative images of the cresyl violet-stained sections show the reduction in lesion volume observed in S100B^−/− samples. Stereological assessment of surviving neurons using unbiased stereological techniques was performed in the cortex (C), thalamus (D), and cornu ammonis 1 (CA1) (E) subregion of the hippocampus at 28 days after injury. TBI resulted in significant neuronal cell loss in the cortex (*P < 0.05, vs. sham), thalamus (*P < 0.05 or **P < 0.01, vs. sham), and CA1 hippocampal subregion (**P < 0.01, vs. sham). S100B^−/− mice did not show any improvement in neuronal survival in all the assessed brain regions. Analysis by one-way analysis of variance followed by Tukey's post-hoc test. (Mean ± s.e.m.; n = 4 to 7/group).

Stereological assessment of the surviving neurons (n = 4 to 7/group) was performed in the cortex (Figure 2C), thalamus (Figure 2D), and CA1 (Figure 2E), CA2/3 and DG (data not shown) subregions of the hippocampus at 28 days after injury. TBI resulted in significant neuronal cell loss in the cortex (Figure 2C, P < 0.05, vs. sham), thalamus (Figure 2D, P < 0.01 or 0.05, vs. sham), and CA1 hippocampal subregion (Figure 2E, P < 0.01, vs. sham). Interestingly, S100B^−/− mice did not show any improvement in neuronal survival in all the assessed brain regions.

Brain injury results in a switch in microglial phenotype from a quiescent form displaying ramified cellular morphologies to more activated forms displaying hypertrophic or bushy morphologies (Figure 3A), with the latter being the most reactive. Stereological assessment of microglial cell number and activation phenotype (n = 4 to 7/group) was performed in the cortex at 7 and 28 days after TBI. S100B^−/− mice (37,071.59 ±5,518.46 counts/mm³) showed statistically significant increases in the numbers of ramified microglia on 28 days after injury (Figure 3B, P < 0.01, vs. sham; P < 0.05, vs. S100B^+/+) when compared with sham (11,219.28± 1,320.44 counts/mm³) and injured S100B^+/+ mice (14,691.67 ±2,331.77 counts/mm³). TBI resulted in significant microglial activation at 7 days after injury (P < 0.05, vs. sham, 41,202.63 ±7,785.17 counts/mm³) followed by sustained increases in the numbers of activated (hypertrophic and bushy) microglia at 28 days (P < 0.001, vs. sham, 70,259.36 ±8,821.79 counts/mm³) when compared with sham (Figure 3B). S100B^−/− mice showed significant attenuation in the numbers of activated microglia at 28 days (P < 0.05, vs. S100B^+/+, 34,325.9 ±5,965.02 counts/mm³) when compared with S100B^+/+ mice.

Figure 3.

S100B^−/− mice had reduced microglial activation in the injured cortex after traumatic brain injury (TBI). Stereological assessment of microglial cell number and activation phenotype was performed in the cortex at 7 and 28 days after TBI. (A) Representative images of the Iba-1-stained sections show morphologic features of ramified and hypertrophic and bushy (activated) microglia. (B) S100B^−/− mice showed statistically significant increases in the numbers of ramified microglia on 28 days after injury (**P < 0.01, vs. sham; ⁺P < 0.05, vs. S100B+^/+, #P < 0.05, vs. S100B^−/− at 7 days) when compared with sham and S100B^+/+ mice (at 28 days) and S100B^−/− mice at 7 days. TBI resulted in significant microglial activation at 7 days after injury (*P < 0.05, vs. sham) followed by sustained increases in numbers of activated microglia at 28 days (***P < 0.001, vs. sham) when compared with sham. S100B^−/− mice showed significant attenuation in the numbers of activated microglia at 28 days (⁺P < 0.05, vs.S100B^+/+) when compared with S100B^+/+ mice. Analysis by one-way analysis of variance followed by Tukey's post-hoc test. (Mean ± s.e.m.; n = 4 to 7/group).

Systemic Treatment With a Neutralizing S100B Antibody Provides Neuroprotection

Behavioral outcomes. Functional assessment of fine motor co-ordination was performed at various time points after injury (n = 8 to 14/injured group, n =6 to 7/sham group) using a beam walk test, and the results for the number of foot faults were statistically analyzed by two-way (injury/sham and treatment) repeated-measures (measures/time) ANOVA followed by Tukey's post-hoc test. The interaction of “injury/sham × PIDs” (F(4,245) = 19.939, P < 0.001) and “injury/sham × treatment” (F(2,245) = 17.680, P< 0.001) was statistically significant, and TBI induced significant sensorimotor impairments at all time points when compared with sham-injured mice (Figure 4A; P< 0.001, vs. sham). Anti-S100B IgG-treated mice exhibited significant improvements in sensorimotor performance at 7, 14, 21, and 28 days (P < 0.05, vs. vehicle and IgG control) after injury when compared with vehicle- and IgG control-treated mice.

Figure 4.

Systemic administration of a neutralizing S100B antibody improved functional outcomes following traumatic brain injury (TBI). (A) TBI induced significant sensorimotor impairments at all time points when compared with sham-injured mice in the beam walk test (***P < 0.001, vs. sham). Anti-S100B immunoglobulin G (IgG)-treated mice exhibited significant improvements in sensorimotor performance at 7, 14, 21, and 28 days (⁺P < 0.05, vs. vehicle; *P < 0.05, vs. IgG control) after injury when compared with vehicle-treated and IgG control-treated mice. Analysis by repeated measures two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. (B) TBI-induced deficits in retention memory (P < 0.001, vs. sham) in the novel object recognition task were significantly attenuated by systemic administration of anti-S100B IgG (⁺⁺P < 0.01, vs. vehicle); data expressed as Discrimination Index analysis by one-way ANOVA followed by Tukey's post-hoc test (mean ± s.e.m.; n = 8 to 14/injured group, n = 6 to 7/sham group).

Spatial learning and reference memory were assessed using the acquisition phase and probe trial of the MWM test, respectively. TBI resulted in spatial learning impairments on PID 17 (data not shown; P < 0.001, vs. sham) in the acquisition phase of MWM test. In contrast, our data from the probe trial of the MWM test indicated that injured animals did not spend significantly greater amount of time in the target quadrant when compared with the sham groups (data not shown), and there were no statistically significant differences between the three injured groups. Anti-S100B IgG-treated mice failed to show improvements in cognitive performance in the MWM test when compared with vehicle- or IgG control-treated mice (data not shown).

Retention or intact memory was evaluated using the NOR test and statistically analyzed by one-way ANOVA followed by Tukey's post-hoc test (F(5,49) = 0.8705;P < 0.0001)). TBI-induced cognitive deficits (P < 0.001, vs. sham) in the NOR task were significantly attenuated by systemic administration of anti-S100B IgG (Figure 4B; P < 0.01, vs. vehicle).

Neuropathologic changes. TBI-induced lesion volume (n =5 to 7/group) was estimated by unbiased stereological techniques. Histologic assessment showed that vehicle-treated mice developed a large lesion following TBI (Figures 5A and 5B, 10.74± 0.62 mm³), whereas anti-S100B IgG treatment resulted in a significant reduction in lesion size (P < 0.001, vs. vehicle;P < 0.05, vs. IgG control, 6.36±0.48 mm³). Interestingly, systemic administration of IgG control significantly reduced the lesion volume (P < 0.01, vs. vehicle, 8.09 ±0.63 mm³).

Figure 5.

Systemic administration of a neutralizing S100B antibody reduced lesion volume, improved neuronal survival, and attenuated microglial activation in the cortex after traumatic brain injury (TBI). (A) Histologic assessment using unbiased stereology showed that vehicle-treated mice developed a large lesion following TBI, whereas anti-S100B immunoglobulin G (IgG) treatment resulted in significant reduction in lesion size (***P < 0.001, vs. vehicle). Systemic administration of IgG control significantly reduced the lesion volume (**P < 0.01, vs. vehicle, ⁺P < 0.05, vs. anti-S100B IgG treatment). Analysis by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (mean ±s.e.m.; n = 5 to 7/group). (B) Representative images of the cresyl violet-stained sections show the reduction in lesion volume observed in anti-S100B IgG-treated samples. (C) Stereological assessment of surviving neurons was performed using unbiased stereological techniques in the cortex at 28 days after injury. TBI resulted in significant neuronal cell loss in the cortex (**P < 0.01, vs. sham). Systemic administration of anti-S100B IgG significantly improved neuronal survival in the cortex (⁺P < 0.05, vs. vehicle) when compared with vehicle-treated samples. Analysis by one-way ANOVA followed by Tukey's post-hoc test (mean ±s.e.m.; n = 5/group). (D) Stereological assessment of microglial cell number and activation phenotype was performed in the cortex at 7 and 28 days after TBI. No statistically significant increases in the numbers of ramified microglia were observed on 7 and 28 days after injury. TBI resulted in significant and sustained increases in activated microglia at 7 days after injury followed by sustained increases at 28 days (***P < 0.001, vs. sham). Systemic administration of anti-S100B IgG significantly attenuated microglial activation at 7 days after injury (⁺⁺⁺P < 0.001, vs. vehicle; (^***P < 0.001, vs. IgG control) when compared with vehicle- and IgG control-treated samples Furthermore, anti-S100B IgG continued to show significant reduction in the numbers of activated microglia at 28 days postinjury (⁺P < 0.05, vs. vehicle) when compared with vehicle-treated samples. Analysis by one-way ANOVA followed by Tukey's post-hoc test (mean ± s.e.m.; n = 5/group).

Stereological assessment of surviving neurons (n = 5/group) was performed in the cortex (Figure 5C), thalamus, and CA1, CA2/3, and DG subregions of the hippocampus (data not shown) at 28 days after injury. TBI resulted in significant neuronal cell loss in the cortex (Figure 5C, P < 0.01, vs. sham), thalamus (data not shown), and all hippocampal subregions (data not shown). Systemic administration of anti-S100B IgG significantly improved neuronal survival in the cortex (Figure 5C; P < 0.05, vs. vehicle, 359,852 ± 54,494.4 counts/mm³) when compared with vehicle-treated samples (173,830.3 ±40,864.82 counts/mm³). However, anti-S100B IgG treatment did not attenuate TBI-induced neuronal cell loss in the thalamus as well as in the hippocampus (data not shown).

Stereological assessment of microglial cell number and activation phenotype (n = 5/group) was performed in the cortex at 7 and 28 days after TBI (Figure 5D). No statistically significant increases in the numbers of ramified microglia were observed on 7 and 28 days after injury (Figure 5D). TBI resulted in significant and sustained increases in activated microglia at 7 days after injury followed by sustained increases at 28 days (Figure 5D;P < 0.001, vs. sham). Systemic administration of anti-S100B IgG significantly attenuated microglial activation at 7 days after injury (P < 0.001, vs. vehicle and IgG control, 28,571.64 ±5,702.16 counts/mm³) when compared with vehicle- (74,305.19 ±7,811.68 counts/mm³) and IgG control-treated (69,807.02 ± 8,947.65 counts/mm³) samples. Furthermore, anti-S100B IgG continued to show significant reduction in the numbers of activated microglia at 28 days after injury (P < 0.05, vs. vehicle, 28,727.6 ±3901.79 counts/mm³) when compared with vehicle-treated (53,348.67 ±4,536.09 counts/mm³) samples.

AGER in the brain after TBI. Proximity ligation assays were used to determine whether S100B activation with AGER contributed to the inflammatory response in TBI. S100B-AGER complexes were not detectable in any region of the TBI or sham brain at 7 days after injury (Figure 6), despite the fact that both S100B and AGER antigens were independently detected in these specimens. Our inability to detect S100B-AGER complexes was not due to technical issues as these complexes were readily discernible in a positive control (Figure 6), and previous studies confirmed the suitability of the primary antibodies for immunohistochemistry (data not shown).

Figure 6.

S100B–AGER complexes are not detectable in traumatic brain injury (TBI). Representative micrographs of ipsilateral cortical regions from TBI/sham sections 7 days after injury and positive/negative control sections processed for proximity ligation assays using S100B+AGER primary antibodies (brown) and counterstained with hematoxylin to visualize nuclei (blue).

S100B does not Influence Key Microglial Activation Markers In Vitro

To determine whether S100B can modulate microglial activation, we examined its effects on NO release and/or ROS production, classic markers of activation using two in vitro microglia models. BV2 microglia were cultured in 96-well plates, pretreated with S100B (1 μmol/L) for 1 hour before and subsequently stimulated by LPS or IFNγ for a further 24 hours. LPS (0.1 to 1μg/ml) and IFNγ (0.2 to 20 ug/ml) significantly increased NO release. We observed no statistically significant changes in NO production in the corresponding samples pretreated with S100B (Figures 7A and 7B). We also evaluated the effects of S100B on activation of primary microglia. These cells were cultured in 96-well plates, pretreated with S100B for 1 hour before and subsequently stimulated by IFNγ for a further 24 hours. IFNγ (0.2 to 20 μg/ml) significantly increased NO release and ROS production. We observed no statistically significant changes in either outcome in corresponding samples pretreated with S100B (Figures 7C and 7D).

Figure 7.

S100B does not influence microglial activation markers in vitro. Lipopolysaccharide (LPS) (A) and interferon-gamma (IFNγ) (B) stimulation in BV2 microglia significantly increased nitric oxide (NO) production at 24 h (***P < 0.001, ****P < 0.0001 vs. control). Pretreatment of cells with S100B (1 μm) did not significantly attenuate LPS- or IFNy-stimulated increases in NO production. Analysis by oneway analysis of variance (ANOVA) followed by Tukey's post-hoc test (mean ±s.d.; n = 6/group). IFNγ stimulation in primary mouse microglia significantly increased NO (C) and reactive oxygen species (ROS) (D) production at 24 h (***P < 0.001, ****P < 0.0001 vs. control). Pretreatment of cells with S100B (1 um) did not significantly attenuate IFNγ-stimulated increases in NO or ROS production. Analysis by one-way ANOVA followed by Tukey's post-hoc test (mean ± s.d.; n = 3/group).

DISCUSSION

S100B belongs to the S100/calmodulin/troponin C superfamily of proteins that function as calcium-modulated proteins and exert their effects on the CNS in a concentration- and calcium-dependent manner. ²⁴ Extracellular S100B promotes neurite extension outgrowth and enhances cell maintenance in cultures of embryonic chick cerebral cortex neurons ²⁵ and stimulates proliferation of primary rat astrocytes. ²⁶ Increased intracellular calcium by S100B protein has been reported to decrease neuronal cell death and mitochondrial dysfunction in rat hippocampal neurons may provide neuroprotection for CNS neurons at nanomolar concentrations. In contrast, other studies have reported neurotoxic, particularly neuroinflammatory effects of S100B, via activation of iNOS-causing astrocytic death at micromolar concentrations. ²⁷ S100B has long been considered an important marker of brain injury after TBI, with elevated serum levels being correlated with poor outcomes both experimentally and clinically.^14,15 Multiple experimental TBI models have shown significant increases in S100B levels in the brain early after TBI ²⁸ and persisting up to 5 days.^29,30 Intraventricular infusion of S100B resulted in a significant improvement in memory function after lateral fluid percussion-induced brain injury, ²⁹ but higher serum levels of S100B were correlated with impaired cognitive performance. The latter finding is in agreement with many clinical observations and was thought to reflect activation of secondary injury mechanisms.

There is a transient increase in blood–brain barrier permeability after CCI in rodents, ³¹ which may be associated with functional changes in the blood–brain barrier rather than mechanical disruption of cerebrovascular barrier. Blood–brain barrier changes and permeability to albumin and other high-molecular weight proteins has been observed within 4 to 6 hours after TBI and persists for several days. This may be due to increased paracellular permeability of endothelial barrier associated with changes in expression, distribution, and function of tight-junction proteins and from modified transport of pinocytic vesicles across blood-brain barrier. Several experimental studies have demonstrated that there is a surge in the production of pro-inflammatory molecules, including S100B ³² by brain parenchymal cells. Several

studies have shown that overproduction of S100B by activated astrocytes after brain injury further increases microglial and astrocyte activation, as well as neuronal cell death. ⁹ Release of S100B can contribute to the generation of pro-inflammatory factors such as iNOS, TNF-α, IL-1β, and IL-6 that worsen neuroinflammation and may contribute to long-term neurologic dysfunction. ⁹ The objective of our study was to further examine the role and potential mechanisms by which S100B can modulate secondary injury. We evaluated the therapeutic potential of S100B inhibition via genetic as well as pharmacological means against TBI-induced microglial activation and behavioral deficits using the well-established CCI mouse model, which recapitulates certain pathophysiologic features of clinical TBI and results in long-term neurologic deficits.^3,18,21 In our study, S100B knockouts or delayed systemic administration of a neutralizing S100B antibody IgG were used to inhibit S100B. Anti-S100B IgG was administered systemically at two delayed time points of 24 hours and 14 days after TBI to simulate a clinically relevant treatment paradigm.

CCI led to long-term behavioral impairment in terms of sensorimotor, spatial, and retention memory functions. CCI-induced sensorimotor deficits were significantly reduced by systemic administration of a neutralizing S100B antibody, which also significantly reduced lesion volume as compared with untreated injured animals. The extent of locomotor impairment after injury to the sensorimotor cortex is often correlated with lesion size. ²¹ However, knocking out S100B reduced lesion volume but did not cause significant improvements in sensorimotor outcomes. Interestingly, treatment with an IgG control after TBI reduced the size of the lesion. The latter finding is consistent with previous studies showing that IgG treatment can improve function after experimental TBI, possibly reflecting its antiinflammatory actions.^33,34 The non-specific IgG effect on the reduction of lesion volume could likely be attributed to an inflammatory or acute phase reaction to the IgG that either counteracts or modulates TBI-induced inflammation in favor of repair or recovery. There is also a possibility of the oncotic colloidal pressure associated with hemodynamic changes and/or edema after TBI being altered after control IgG administration.^33,34 However, the molecular mechanisms underlying this effect have not been established to date and need to be investigated.

To further evaluate treatment effects, neuronal cell loss was quantitatively assessed in the cortex and thalamus using unbiased stereological techniques. CCI caused neuronal loss in both cortex and thalamus. Acute S100B inhibition with a neutralizing S100B antibody, but not IgG treatment, significantly improved neuronal survival in the cortex but not in the thalamus. In contrast, chronic S100B inhibition using genetic intervention did not cause a statistically significant attenuation of CCI-induced neuronal loss in any of the assessed brain regions, though we observed a trend towards increased neuronal survival in the cortex of injured S100B^−/− mice. Improvement in neuronal survival in the cortex may not be directly correlated with the reduction in the size of the cortical lesion, as the cortical neuronal counts represent changes surrounding the lesion and not just at the site of the lesion, whereas the lesion volume is determined by the missing tissue at the site of the lesion.

To determine the effect of S100B inhibition on memory function, different cognitive tasks were used. Improved cognitive performance in the MWM of spatial learning and reference memory serves as a potential indicator of reduced hippocampal damage. CCI induced significant cognitive deficits in acquisition phase of the MWM task, indicating impairment in spatial learning (data not shown). In contrast, our data from the probe trial of the MWM test after both interventions indicated that injured animals did not spend significantly greater amount of time in the target quadrant when compared with sham groups and therefore did not show statistically significant impairment in reference memory function in this test. Neither genetic ablation nor neutralizing antibodies altered MWM performance after CCI (data not shown). Improved memory function in the MWM test is demonstrated by a strong correlation between cognitive improvement and increased neuronal survival in the CA3 and DG hippocampal subregions.^18,21 CCI caused significant neuronal cell loss in CA1, CA2/3, and DG hippocampal subregions, which was not modified by S100B inhibition. Retention memory can be assessed using the NOR test, which serves as a measure of object discrimination/exploration based on retention memory function as well as locomotor activity. Performance in the NOR test reflects both cortical and hippocampal function.^18,21 CCI caused significant impairment in retention memory based on object discrimination/exploratory behavior. Either knocking out S100B or administration of a neutralizing antibody significantly improved functional outcomes in the NOR test. Together, these findings suggest that effects of S100B inhibition on retention memory function in the NOR test likely reflect neuroprotective effects in the cortex.

Chronic inflammation after CNS trauma may provide a mechanistic link between early and chronic neurodegeneration. ³ Previous studies have suggested that sustained microglial activation after TBI may contribute to chronic neuronal cell loss and associated neurologic dysfunction.^3,4,21 We have previously demonstrated using stereological assessment and 3D reconstruction analysis in multiple models of TBI^3,4,21 that the process of microglial activation after TBI is slow to start as indicated by the low numbers of microglia displaying activated (hypertrophic and bushy) morphologies at 24 hours, followed by a statistically significant elevation in the numbers of activated microglia at 7 days after injury, which is the peak in activation in these models. Although these numbers decline at later time points compared with peak levels at 7 days after injury, microglia are chronically activated up to 12 months after injury, such that there are significantly increased numbers of activated microglial cells at 12 months after injury when compared with sham-injured control levels. ³⁵ Interestingly, our previous pharmacological intervention studies using cell cycle inhibitors demonstrated that the 7- and 28- day time points were ideal to evaluate treatment effects on posttraumatic microglial activation, both at the peak and at a more chronic time point.^4,21 Therefore, based on our previously published findings on the time course and response of an intervention to microglial activation after TBI, we performed a quantitative assessment of microglial activation after TBI at 7 and 28 days, respectively. Microglial activation status was based on morphologic features using unbiased stereology, as described previously.^3,21 CCI significantly increased the numbers of activated microglia at 7 days when compared with sham controls, with increases sustained at 28 days. Knocking out S100B also significantly attenuated the numbers of activated microglia and increased the numbers of ramified microglia at 28 days. Neutralizing antibodies significantly attenuated the numbers of activated microglia at 7 and 28 days after CCI. Unlike the lesion size, treatment with control IgG did not cause attenuation of cortical microglial activation after TBI. The administration of control IgG did not attenuate sensorimotor deficits, improve neuronal survival, and reduce microglial activation; in contrast, these improved outcomes were observed with anti-S100B IgG treatment, suggesting that the non-specific IgG component of the neutralizing S100B antibodies contributes to only a small part of the neuroprotection provided by anti-S100B IgG treatment. Future studies will provide insight into the non-specific IgG effect and will highlight the specific inhibitory effect of S100B.

Our data strongly implicate a role for S100B protein in the pathophysiology of TBI and demonstrate that inhibition of S100B limits microglial activation and reduces the extent of trauma-induced neurologic damage and associated functional deficits. Previous studies using primary microglia from newborn rat cortex or BV-2 microglial cell lines have demonstrated that micromolar to nanomolar concentrations of S100B cause IFNγ-induced expression of iNOS as well as NO secretion.^10,36 In contrast, our studies using BV2 and primary microglial cultures stimulated by LPS and/or IFNγ showed no effects of S100B on key markers of microglial activation, such as NO release or ROS production. Furthermore, it has been proposed that S100B may act through AGER and subsequently Toll-like receptors to induce inflammatory responses.^7,36 Although our proximity ligation assays do not support a direct interaction in the brain between S100B and AGER at early time points after TBI in our model, the relationships between S100B and AGER are complex. The AGER-transducing activity may not have a significant role in S100B-stimulated NO production by microglia, but the AGER extracellular domain may be important for concentrating S100B on the BV-2 cell surface. ³⁶

Studies have demonstrated that S100B-stimulated NO release from the microglia may be dependent on the density of AGER molecules on microglial surface and activation of pathways such as p38 MAPK (mitogen-activated protein kinase) that result in production of ROS. ³⁶ Extracellular S100B can promote AGER-dependent AP1 and nuclear factor (NF)-kB-dependent gene transcription of cytokines, chemokines, and iNOS, as well as AGER-independent NO production in microglia. ²⁷ In astrocytes, extracellular S100B can activate AGER/Rac-1/Cdc42, AGER/Erk-Akt, and AGER/NF-KB pathways. ³⁷ Extracellular S100B can also facilitate neurodegeneration via AGER/MAPK/cyclinD1-CDK4 activation and cell cycle re-entry ³⁸ as well as AGER/NF-KB activation. ³⁹ Furthermore, S100B has been shown to stimulate the production of TNF-α in the microglia through concurrent activation of Rac1-NF-KB module, Rac1-JNK-AP-1 module, and potential involvement of ERK1/1-NF-KB and p38MAPK-NF-KB modules.^7,40 TNF-α has an important role in S100B release from the astrocytes, suggesting that TNF-α might elevate the extracellular concentration of S100B favoring activating effects of S100B on microglia.^7,40 Collectively, our data and previously demonstrated mechanisms indicate that S100B contributes to the activation and progression of neuroinflammatory processes following TBI. However, S100B inhibition may directly or indirectly attenuate neuroinflammation after TBI, and future studies are needed to determine the mechanisms of S100B-mediated neuroinflammatory effects.

In conclusion, our study indicates that S100B has a role in TBI-induced microglial activation and associated neurologic dysfunction. Furthermore, our data support the potential of S100B as a therapeutic target for treatment of TBI. The mechanisms by which S100B contributes to the pathobiology of TBI do not appear to involve direct S100B-GER interactions. Further studies are needed to elucidate the specific pathways underlying S100B-mediated neuroinflammatory and related neurodegenerative actions after TBI.