Deficiency in interleukin-18 ameliorated glial activation and neuroinflammation after traumatic brain injury in mice

Background: Neuroinflammation is recognized as one of the main pathological mechanisms of secondary injury caused by traumatic brain injury (TBI). It has been reported that interleukin (IL)-18 is expressed in glial cells and involved in the regulation of neuroinflammation. Further studies have revealed that IL-18 expression is upregulated and may contribute to pathogenesis in the later phases of TBI; however, the mechanism underlying the effect of IL-18 on TBI remains unclear. Our present study assessed the roles of IL-18 in inflammatory and neurodegenerative pathology in mice subjected to TBI. Methods: A controlled cortical impact (CCI) injury model was conducted to mimic TBI, and brains were collected at 3 and 7 days post TBI (dpi). The levels of IL-18 were detected by qRT-PCR and immunofluorescence staining. In addition, neurological severity score (NSS) was used to assess neurological deficits after TBI. Furthermore, neuronal cell death, glial activation, and inflammatory cytokine and chemokine secretion were evaluated in wild-type ( WT ) and Il18-knockout ( Il18 -KO) mice to explore the role of IL-18 in TBI. Results: IL-18 levels were upregulated post TBI, accompanied by reactive glial activation. Il18 deficiency significantly ameliorated glial activation and improved neuronal cell death and neurological deficits. In addition, Il-18 deficiency reduced the TBI-induced M1-like microglia frequency. Interestingly, the levels of all pro- and anti-inflammatory cytokines, including IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL17A, G-CSF, GM-CSF, IFN-γ, and TNF-α, were downregulated in Il18 -KO mice. The deletion of Il-18 attenuated the levels of most chemokines induced by TBI, including CCL2, CCL3, The samples were analyzed using a and two premade kits for 17 cytokines (including IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, G-CSF, GM-CSF, IFN-γ, and TNF-α) and 18 chemokines (including CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL7, CCL8, CCL11/Eotaxin-1, CCL12, CCL19, CCL20, CCL21, CCL22, CXCL1/GRO-α/KC, CXCL2/GRO-β, CXCL10, CXCL12, CXCL13, and CXCL16). Cytokines and chemokines were analyzed using Luminex technology following the manufacturer’s specifications (Bio-Plex Cytokine 23-plex, deoxynucleotidyl transferase UTP nick end labeling; NSS: neurological severity score; G-CSF: Granulocyte-colony stimulating factor; GM-CSF: granulocyte-macrophage colony-stimulating factor; DAMPs: danger-associated molecular pattern; CCL: chemokine (C-C motif) ligand; CXCL: chemokine (C-X-C motif) ligand; CNS: central nervous system.


Background
Traumatic brain injury (TBI) is a major cause of death and disability worldwide and represents a critical public health problem, especially in China [1][2][3]. TBI is defined as brain damage caused by an increased levels of serum IL-18 are associated with the exacerbation of long-term outcomes in TBI patients [21]. These studies suggest that IL-18 may contribute to the repair process after TBI in rodents, which is worth exploring.
In the current study, using a well-characterized controlled cortical impact (CCI) injury model, we assessed the roles of IL-18 in inflammatory and neurodegenerative pathology in genetically engineered Il-18 knockout (Il18-KO) mice. We found that the expression of IL-18 was increased, which was accompanied by robust reactive glial activation. Genetic deletion of Il-18 resulted in a decreased neuroinflammatory response, reduced cell death, and ameliorated neurological deficits. These results show that IL-18 may be an important target in the regulation of TBI-induced neuroinflammation.

Animals
Male C57BL/6 (wild-type, WT) and Il18-KO (Stock No: 004130, purchased from the Jackson laboratory) mice (8-12 weeks; 20-26 g) were used in this study [22]. The mice were housed at constant temperature and humidity under a 12-hour light/dark cycle. Food and water were provided ad libitum.
Il18-KO mice were genotyped by PCR amplification of genomic DNA extracted from tail snips (Fig.   S1a). The Il-18 mRNA level in the mouse brain was also determined by qRT-PCR (Fig. S1b). The protocols for animal experiments were approved by the China Medical University Animal Care and Use Committee.

Traumatic brain injury
Moderate TBI was induced according to a CCI injury model that was described previously with slight modifications [23][24][25]. Briefly, mice were anesthetized, shaved and maintained with 2.5% isoflurane with a nonrebreathing nose cone and passive exhaust system connected to a stereotaxic frame. Body temperature was maintained at 37°C during the surgical procedure. The skull was exposed by making a midline incision, and a craniotomy approximately 4 mm in diameter was made using an electric microdrill on the central aspect of the left parietal bone, with the center of the opening located 2.5 mm posterior to the bregma and 2.5 mm lateral to the sagittal suture. The bone flap was removed without damage to the exposed underlying dura. Any animal with a disrupted dura was omitted from 6 the study and replaced by another littermate. The contusion device consists of a computer-controlled electromagnetic driven impactor, with a 3-mm-diameter tip (Pinpoint PCI3000 Precision Cortical Impactor). The manipulator arm was rotated 20° on the vertical axis, to allow impact flush with the dura. Moderate injury was delivered by using this device to compress the cortex to a depth of 1.0 mm at a velocity of 1.5 m/s and duration of 50 ms. Then, the incision was sutured closed, and the animal was allowed to recover on a heating pad. Sham animals underwent the same procedures (including craniotomy) as injured mice except for the impact protocol.
In this study, mice survived for 3 or 7 days post injury (dpi), depending on group assignment. A total of 48 WT and 36 Il18-KO mice were assessed, and six mice were randomly included in each group.
Investigators were blinded to the treatment groups during experimental assays and data analysis according to published guidelines.

Processing of brain tissue for histology
At 3 and 7 dpi, mice were deeply anesthetized with an overdose of inhaled isoflurane and then transcardially perfused with cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) at 4°C. Brains were dissected and post-fixed with 4% PFA for 24 hours at 4°C. The tissues were then dehydrated with ethanol, cleared in xylene, and paraffin embedded. Serial coronal sections (4 μm) were cut and mounted on glass slides, followed by immunofluorescence staining. Fluoro-Jade C (FJC) staining and terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) staining were performed. Images were acquired with a Leica DMi8 fluorescence microscope (Leica, Germany). Mice harvested for qRT-PCR and Luminex were perfused with cold PBS only, and the ipsilateral cortex was collected.
Positive cells were counted by an investigator blinded to experimental grouping. The regions of interest are shown in Fig. 1a.

Fluoro-Jade C (FJC) staining
Fluoro-Jade C (FJC) is a polyanionic fluorescein derivative that specifically stains all degenerating neurons, regardless of the mechanism of cell death. FJC staining of brain sections was performed according to our published protocol [26]. First, sections were deparaffinized through two 10-minutes incubations with xylene and rehydrated in a graded alcohol series. The sections were then incubated in a 0.06% potassium permanganate solution for 10 minutes, followed by rinsing with distilled water for 2 minutes and incubation in a 0.0001% solution of FJC (Histo-Chem Inc.) dissolved in 0.1% acetic acid.

TUNEL staining
Apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) detection kit (Beyotime, C1086) according to our published protocol [26]. Briefly, sections were deparaffinized with xylene and dehydrated with a graded ethanol series. Then, the sections were digested with proteinase K (Beyotime, ST533) for 15 minutes. After several washes with PBS, the sections were further incubated with 40 µl of the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and FITC-labeled dUTP at 37°C for 2 hours in the dark. Nuclei were stained with 8 DAPI. The apoptotic index (AI), which was defined as the average percentage of TUNEL-positive cells in ten cortical microscopic fields (×400) in each section, was used to demonstrate the extent of brain damage. The final average of the percentage of TUNEL-positive cells in six sections per animal was considered.

Gene expression assessment
To isolate total RNA from the ipsilateral cortex, tissue samples were homogenized in TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The isolated total RNA was precipitated out using isopropanol, washed with ethanol, and then dissolved in DEPC-treated water. Total RNA concentrations were determined using ultraviolet spectrophotometry, with 260/280 ratios of 1.8-2. The PCR profile consisted of 1 cycle at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 20 s. Gene expression was normalized to Gapdh expression, and the relative quantity of mRNAs was analyzed by the ∆∆CT method [27]. All PCR reactions were performed in triplicate.

Neurological severity score (NSS)
A 10-point NSS was used to quantify neurological deficits after TBI as described previously [28]. The score consists of 10 individual tasks, including exit circle, monoparesis/hemiparesis, straight walk, startle reflex, seeking behavior, beam balancing, round stick balancing, beam walk (3 cm), beam walk (2 cm) and beam walk (1 cm). One point is given for failing to perform each of the tasks. A maximal NSS of 10 points thus indicates severe neurological deficits, with failure of all tasks.

Statistical analysis
All statistical analyses were performed with GraphPad Prism 7 software. All data are expressed as the mean ± standard deviation (SD). Two-way ANOVA was performed for comparisons among groups.
Tukey's post hoc analysis was used to determine where differences occurred. p< 0.05 was considered statistically significant.

IL-18 expression is upregulated in the injured ipsilateral cortex after TBI
Studies have previously reported that IL-18 levels increase significantly at 3-7 dpi [17-19], suggesting the involvement of IL-18 in the pathogenic processes of TBI. To assess the expression level of IL-18 in the injured ipsilateral cortex in our study, we determined Il-18 gene expression using qRT-PCR and performed immunofluorescence staining using an antibody specific to IL-18. Il-18 mRNA levels in the injured ipsilateral cortex were increased significantly in the TBI groups compared with the sham groups at 3 and 7 dpi (Fig. 1b). Immunofluorescence staining revealed that IL-18 expression was increased in the injured ipsilateral cortex at 3 and 7 dpi (Fig. 1c). To identify the cell types that expressed IL-18 post TBI, we performed double immunostaining with an anti-IL-18 and a neuronspecific anti-NeuN antibody, astrocyte-specific anti-GFAP antibody, or microglia-specific anti-IBA1 antibody. Double immunofluorescence indicated that IL-18 was mainly colocalized with IBA1+ microglia and NeuN+ neurons, but not with GFAP+ astrocytes, at 7 dpi (Fig. 1d).

Il-18 deficiency ameliorates neurological deficits caused by TBI
To assess the role of IL-18 in neurological deficits, a 10-point NSS was assessed. As shown in Fig. 2, NSS scores were significantly increased in TBI mice compared with sham animals at 3 and 7 dpi in WT and Il18-KO mice (Fig. 2). However, deficiency in Il-18 resulted in significant decreases of NSS at 3 and 7 dpi compared with that in WT mice (Fig. 2).

Deletion of Il-18 attenuates neuronal degeneration and cell death after TBI
To explore the role of IL-18 in neuron survival after TBI, immunostaining for MAP2, a neuron-specific biomarker, was carried out. As shown in Fig. 3, the number of MAP2+ cells was decreased at 3 and 7 dpi in WT and Il18-KO mice compared with sham mice (Fig. 3). More neurons survived in the mice with Il-18 deletion than in the WT mice (Fig. 3).
TBI is associated with severe neurodegeneration. We performed FJC staining to examine the degenerated cells in the ipsilateral cortex after TBI. Increased numbers of FJC+ cells were detected in the cortex at 3 and 7 dpi in WT and Il18-KO mice compared with sham animals (Fig. 4). Deficiency in Il-18 prevented TBI-induced neuronal degeneration, with evidence demonstrating that markedly fewer FJC-positive cells were found post-injury in Il18-KO mice than in WT mice (Fig. 4).
To evaluate apoptotic cell death in the pericontusional area of the ipsilateral cortex, an in situ TUNEL assay was conducted. As shown in Fig. 5, TUNEL+ cells were evident at 3 and 7 dpi in the pericontusional tissue. The apoptotic index (AI) increased significantly in the TBI groups at 3 and 7 dpi in WT and Il18-KO mice compared with that in the sham groups (Fig. 5). Quantitative analysis indicated that mice with Il-18 deletion showed lower AI post trauma than WT animals (Fig. 5b).

TBI-induced astrogliosis is attenuated in Il18-KO mice
Astrocytes and microglia are considered key players in initiating an inflammatory response after injury. To explore the effect of IL-18 on the TBI-induced inflammatory response, we first evaluated astrogliosis in the ipsilateral cortex by GFAP immunostaining at 3 and 7 dpi. After TBI, GFAP+ cells were activated and increased significantly, showing a hypertrophic morphology involving swelling of cell bodies and extension of processes (Fig. 6). The number and percent area of GFAP+ cells increased significantly at 3 and 7 dpi in WT and Il18-KO mice (Fig. 6b, c). Mice with Il-18 deletion exhibited fewer GFAP+ cells and a lower percent area of GFAP+ cells in the injured ipsilateral cortex compared with WT animals at 3 and 7 dpi (Fig. 6b, c). Furthermore, GFAP+ reactive astroglia in WT mice showed bulky and long branches with very few secondary processes, while GFAP+ astroglia emanated slender and relatively short primary processes with numerous secondary processes in mice with Il-18 deletion (Fig. 6a lower panel), suggesting that IL-18 may participate in the activation of astrocytes.

M1-like 'proinflammatory' subtype
Next, due to the contribution of microglia in TBI, we detected activated microglia by immunostaining for the microglia-specific marker IBA1. In the pericontusional tissue of sham mice, a few quiescent microglia were found with small cell bodies and a fine appearance. Microglia/macrophages were rapidly increased, as observed by IBA1 immunostaining, and exhibited a deramified and hypertrophic morphology after TBI (Fig. 7). The number and percent area of IBA1+ cells increased significantly at 3 and 7 dpi in WT and Il18-KO mice (Fig. 7b, c). Mice with Il-18 deletion exhibited fewer IBA1+ cells and a lower percent area of IBA1+ cells in the injured ipsilateral cortex than WT animals at 7 dpi (Fig. 7b,   c).
Microglial polarization has been shown to vary over time and between different TBI models. M1-like 'proinflammatory' and M2-like 'anti-inflammatory' microglia/macrophages are characterized by different gene expression patterns and have differential functions in injury [7,9]. To explore the effect of IL-18 on M1/M2 polarization following TBI, we evaluated the M1 phenotype by CD32 immunostaining and the M2 phenotype by CD206 immunostaining at 7 dpi in the ipsilateral cortex.

Discussion
Increasing evidence suggests that IL-18 plays a pivotal role in brain injury [19,29,30]. However, the underlying mechanism remains unclear. The current study has demonstrated that IL-18 levels are increased and that Il-18 deficiency is neuroprotective in TBI. We observed TBI-induced expression of After TBI, microglia become activated, and the cells number peaks at 7 dpi [41]. Microglia release proinflammatory cytokines, including IL-1β, IL-6, IL-12, and TNF-α, and chemokines, including CCL2, CCL3, CCL5, CXCL10, and CXCL12 [50]. Activated microglia are described by the nomenclature 'M1' and 'M2' [7,9]. Microglia polarize from a resting phenotype into the M1-like phenotype after exposure to danger-associated molecular patterns (DAMPs), free radicals, or proinflammatory cytokines such as