s2fieldsofstudy: Biology Medicine

text:
Placental ischemia increases seizure susceptibility and cerebrospinal fluid cytokines

Eclampsia is diagnosed in preeclamptic patients who develop unexplained seizures and/or coma during pregnancy or postpartum. Eclampsia is one of the leading causes of maternal and infant morbidity and mortality, accounting for ∼13% of maternal deaths worldwide. Little is known about the mechanisms contributing to the pathophysiology of eclampsia, partly due to the lack of suitable animal models. This study tested the hypothesis that placental ischemia, induced by reducing utero-placental perfusion, increases susceptibility to seizures, cerebrospinal fluid (CSF) inflammation, and neurokinin B (NKB) expression in brain and plasma. Pentylenetetrazol (PTZ), a pro-convulsive drug, was injected into pregnant and placental ischemic rats (40 mg/kg, i.p.) on gestational day 19 followed by video monitoring for 30 min. Seizure scoring was blindly conducted. Placental ischemia hastened the onset of seizures compared to pregnant controls but had no effect on seizure duration. Placental ischemia increased CSF levels of IL-2, IL-17, IL-18 and eotaxin (CCL11), had no effect on plasma NKB; however, PTZ increased plasma NKB in both pregnant and placental ischemic rats. NKB was strongly correlated with latency to seizure in normal pregnant rats (R2 = 0.88 vs. 0.02 in placental ischemic rats). Lastly, NKB decreased in the anterior cerebrum in response to placental ischemia and PTZ treatment but was unchanged in the posterior cerebrum. These data demonstrate that placental ischemia is associated with increased susceptibility to seizures and CSF inflammation; thus provides an excellent model for elucidating mechanisms of eclampsia-like symptoms. Further studies are required to determine the role of CSF cytokines/chemokines in mediating increased seizure susceptibility.

Introduction
Preeclampsia is a pregnancy disorder characterized by new-onset hypertension after the 20th week of gestation, accompanied by proteinuria or in the absence of proteinuria, low platelet count, renal insufficiency, impaired liver function, pulmonary edema, or cerebral or visual symptoms (American College of Obstetricians and Gynecologists, 2013). Preeclampsia affects 5-8% of all pregnancies in the United States (Saftlas et al. 1990) and close to 20% of African American pregnancies (Mostello et al. 2002). Currently, early delivery of the fetus and removal of the placenta are the only methods to reverse the disorder (Sibai et al. 2005;Sibai 2006), contributing to increased preterm births. Preeclampsia is a multi-organ disorder, affecting the kidney, liver, and brain. Importantly, of all preeclampsia/eclampsia-related deaths, cerebrovascular events are the cause in~40% of cases (MacKay et al. 2001). Additionally, women with preeclampsia present with neurological symptoms such as headaches, blurred vision, nausea, drowsiness, and seizures (in the case of eclampsia) (Chakravarty and Chakrabarti 2002). Furthermore, women with preeclampsia have increased risk of stroke during pregnancy and postpartum (Tang et al. 2009).
Preeclampsia can quickly progress to eclampsia, diagnosed in preeclampsia patients without a history of epilepsy, presenting with new onset seizures or convulsions during pregnancy or postpartum. According to the Preeclampsia Foundation, eclampsia is one of the top five causes of maternal and infant mortality and morbidity and is responsible for approximately 13% of all maternal deaths worldwide (Nour 2008). Importantly, eclampsia has been described as the highest predictor of postpartum acute cerebrovascular disease (Hovsepian et al. 2014). Despite these statistics, the mechanisms contributing to the onset of eclampsia symptoms are not clear due to immediate intervention in the form of delivery following stabilization in women with eclamptic episodes as well as lack of suitable animal models.
Studies describing potential animal models of eclampsia have been recently described in which animal models of preeclampsia are used to explore susceptibility to druginduced seizures. For example, one study induced preeclampsia-like symptoms by administering lipopolysaccharide to pregnant rats followed by pentylenetetrazol (PTZ) treatment to induce seizures and found that lipopolysaccharide-treated animals had reduced threshold to seizures (Huang et al. 2014). Another study used placental ischemia coupled with a high cholesterol diet followed by PTZ injection and reported reduced seizure threshold and increased susceptibility to seizures in response to placental ischemia and high cholesterol diet treatment (Johnson et al. 2014). While these studies provide evidence for increased seizure susceptibility in animal models of preeclampsia, it is not known whether placental ischemia by itself can induce increased sensitivity to PTZinduced seizures.
The placental ischemia model of preeclampsia mimics the clinical condition very well. For example, like preeclamptic patients, placental ischemic rats have increased arterial blood pressure, with or without proteinuria (Alexander et al. 2001), increased inflammatory cytokines (LaMarca et al. 2005) (Gadonski et al. 2006), and increased anti-angiogenic factors (Gilbert et al. 2009) (Gilbert et al. 2007). Additionally, placental ischemic rats have impaired cerebral blood flow autoregulation and increased blood-brain barrier permeability (Warrington et al. 2014) along with impaired cerebrovascular myogenic tone and edema (Ryan et al. 2011). While placental ischemia shares numerous charac-teristics of preeclampsia in patients, whether placental ischemia increases seizure susceptibility and whether it can be utilized as a potential animal model of eclampsia is not known.
Neurokinin B (NKB) is a neuropeptide expressed widely in the central nervous system and peripheral nervous system. It belongs to the family of neuropeptides called tachykinins that includes neurokinin A and Substance P. NKB is found in neurons and immune cells in the nervous system but have also been shown to be expressed in placenta (Page and Lowry 2000; in the outer syncytiotrophoblasts near the maternal circulation at term. Based on literature showing increased NKB in plasma from preeclampsia women D'Anna et al. 2004;Liu et al. 2009), I hypothesized that NKB will be increased in the circulation and placentas of placental ischemic rats.
This study assessed whether placental ischemic rats have increased susceptibility to pentylenetetrazol (PTZ)induced seizures compared to normal pregnant rats. As a potential mechanism, levels of 27 cytokines/chemokines in the cerebrospinal fluid were measured. Neurokinin B (NKB) was measured in the circulation, placenta, and brain in response to placental ischemia and PTZ injection. This is the first study to assess sensitivity to seizures in response to placental ischemia and changes in cerebrospinal fluid cytokines/chemokines in an animal model of preeclampsia.

Animals
Timed pregnant Sprague-Dawley (CD) rats were obtained from Charles Rivers Laboratories and arrived at the Lab Animal Facilities at the University of Mississippi Medical Center on gestational day 11. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center. The rats were maintained on a 12 h light/12 h dark cycle and fed standard rodent chow and water ad libitum.
Reduced uterine perfusion pressure model of placental ischemia Placental ischemia was induced as previously described (Warrington et al. 2014). Briefly, silver clips were surgically inserted around the abdominal aorta and each of the ovarian arteries that branch from the uterine artery on day 14 of gestation ). This method leads to a reduction of blood flow to the utero-placental unit by~40% in pregnant rats .

Seizure induction and monitoring
On gestational day 19, normal pregnant and placental ischemic rats were injected intraperitoneally using 40 mg/kg PTZ (a sub-convulsive dose) and then placed in observation cages. Rats were then video recorded for 30 min. Seizure activity was then determined using the Racine scale by a technician in the Animal Behavior Core Facility, blinded to the treatment. Five stages of intensity were used based on the behavior of the mice: Stage 1: mouth and facial movements, Stage 2: head nodding, Stage 3: forelimb clonus, Stage 4: tonic-clonic seizures, and Stage 5: generalized tonic-clonic seizures characterized by rearing and falling. In addition to the types/stages of seizures observed, latency to first seizure and duration of seizures were also recorded.

Collection of cerebrospinal fluid
On gestational day 19, following completion of seizure monitoring, rats were anesthetized and secured on a stereotaxic frame and cerebrospinal fluid was obtained via the cisterna magna using a butterfly needle and syringe. CSF samples were flash frozen in liquid nitrogen and stored at À80°C until processing.

Determination of brain water content
On gestational day 19, following the collection of CSF samples, rats were euthanized under isoflurane anesthesia and brains were quickly removed and processed as described previously (Warrington et al. 2014). Briefly, the brains were hemisected along the midline and separated into anterior and posterior cerebrum. The junction of the middle cerebral artery to the circle of Willis was used as a landmark for dissection. Tissue rostral of the middle cerebral artery was designated as the anterior cerebrum and tissue caudal of the middle cerebral artery, excluding the cerebellum was considered posterior cerebrum. The brain regions were then weighed and subjected to drying in an oven at 60°C for 72 h, after which dry weight was obtained. Brain water content was then calculated as a percentage: [(wet weight À dry weight)/wet weight] 9 100.

Measurement of CSF cytokine/chemokine concentration
Cerebrospinal fluid (CSF) samples were thawed and run on the rat cytokine/chemokine magnetic bead panel (Milliplex MAP kit, Billerica, MA) which included 27 cytokines/chemokines. Manufacturer's directions were followed and CSF samples were assayed undiluted. Cytokine/chemokine concentration was determined from a standard curve generated for each analyte.

Assessment of Neurokinin B concentration
Neurokinin B was analyzed in the plasma, placentas, and brains of placental ischemic rats and rats were subjected to seizures using a commercially available Rat Neurokinin B EIA kit following manufacturer's directions. Brains were collected from rats and dissected into anterior and posterior cerebrum, and flash frozen in liquid nitrogen. Anterior and posterior cerebral regions were assessed separately because previous studies demonstrate that placental ischemia preferentially affects the anterior cerebrum with no change in the posterior cerebrum (Warrington et al. 2014(Warrington et al. , 2015. Increases in brain water content and blood-brain barrier permeability were selective to the anterior cerebrum (Warrington et al. 2014). Neurokinin B concentrations were calculated using an equation generated from the standard curve as per the manufacturer's directions.

Statistical analysis
The significance of mean values in the normal pregnant and placental ischemic groups was determined using an unpaired t-test. Differences in neurokinin B concentration were determined using Two-Way ANOVA followed by Fisher's LSD test. Comparisons were considered statistically significant if P < 0.05.

Results
Placental ischemia leads to decreased latency to seizures with no change in seizure duration Placental ischemic rats had reduced latency to seizure (264.5 AE 78.7 sec) compared to normal pregnant rats (835.7 AE 246.6 sec; P < 0.05) (Fig. 1A), but had a similar duration of seizures regardless of the seizure classification (P > 0.05). The duration of stages 1-4 seizures was 31.29 AE 6.81 sec in the normal pregnant group compared to 57.25 AE 32.38 sec in placental ischemic rats (Fig. 1B, P = 0.238). The duration of stage 5 seizures was 15.33 AE 3.52 sec in the normal pregnant rats and 54.43 AE 43.96 sec in the placental ischemic rats (Fig. 1C, P = 0.216). Total duration of seizures was 44.43 AE 10.06 sec in the pregnant group and 102.9 AE 71.2 sec in the placental ischemic group (Fig. 1D, P = 0.231).

Placental ischemia leads to increased cytokines/chemokines in the cerebrospinal fluid
Placental ischemia led to increased cerebrospinal fluid levels of four cytokines/chemokines ( Page 3 P = 0.004). Interleukin-17, a proinflammatory cytokine was also increased in the cerebrospinal fluid of placental ischemic rats (20.2 AE 3.7 pg/mL vs. 12.9 AE 1.2 pg/mL in pregnant rats; P = 0.034). Interleukin-18 increased from 40.1 AE 5.1 pg/mL in normal pregnant rats to 60.9 AE 8.8 pg/mL in placental ischemic rats; P = 0.029. Lastly, the proinflammatory cytokine, interleukin-2 was significantly increased in the placental ischemic group (59.5 AE 9.5 pg/ mL) compared to the normal pregnant group (40.7 AE 4.8 pg/mL; P = 0.046). There was a tendency for increased levels of fractalkine (P = 0.069), interferon gamma (P = 0.057), IL-10 (P = 0.071), and VEGF (P = 0.071). The levels of all 27 cytokines/chemokines in the cerebrospinal fluid in response to placental ischemia are shown in Table 1.

Placental ischemia and PTZ increases brain water content
Brain water content increased significantly in response to placental ischemia (Fig. 2, P = 0.012) as well as PTZ treatment (P = 0.014). Placental ischemia increased brain water content from 78.4 AE 0.1% in the pregnant group to 78.8 AE 0.1% (P < 0.05). PTZ increased brain water content in the pregnant group to 78.8 AE 0.1% (P < 0.05) and did not change the brain water content in the placental ischemic group (78.9 AE 0.1%).

PTZ increases circulating neurokinin-B levels
There was a main effect of PTZ treatment on circulating neurokinin-B levels ( Fig. 3A, P < 0.001). Placental ische-mia had no effect on plasma neurokinin-B levels (40.2 AE 3.4 pg/mL in pregnant untreated rats vs. 45.5 AE 3.0 pg/mL in untreated placental ischemic rats). PTZ increased neurokinin B levels in the pregnant rat to 64.0 AE 4.7 pg/mL (P < 0.05) and placental ischemic rats to 57.9 AE 5.3 pg/mL (P < 0.05). The level of neurokinin B present in the circulation was negatively correlated to the latency to seizures in the pregnant group (slope = À42.19; R 2 = 0.877; P = 0.019); however, there was no correlation between the circulating neurokinin B levels and latency to seizure in the placental ischemic group (slope = À3.06; R 2 = 0.019; P = 0.826). Neither placental ischemia nor PTZ had an effect on the expression of neurokinin B protein levels in the placenta (data not shown). However, placental ischemia induced a significant reduction in neurokinin B in the anterior cerebrum (P = 0.034; Fig. 3C) but no change in the posterior cerebrum (P > 0.05; Fig. 3D). PTZ treatment led to a reduction in neurokinin B expression in both the normal pregnant and placental ischemic rats in the anterior cerebrum (P = 0.001), but had no effect in the posterior cerebrum (P > 0.05).

Discussion
Eclampsia is diagnosed when the manifestation of seizures or unexplained coma occurs during pregnancy or the postpartum period in a pregnant or preeclamptic woman with no previously diagnosed seizure disorder. The mechanisms contributing to seizures in eclampsia patients are not known. This study presents the rat model of placental ischemia combined with PTZ treatment as a potential model of eclampsia. Results indicate that placental ischemia led to a reduced latency or hastened onset of first seizure in response to a subconvulsive dose of PTZ. While it is well established that preeclampsia and the rat model of placental ischemia are characterized by increased levels of circulating and placental inflammatory cytokines, it is not known whether cerebrospinal fluid levels of cytokines/chemokines are altered. Because the cerebrospinal fluid has direct contact with the brain and surface vessels, increased cytokines in the cerebrospinal fluid can directly affect brain signaling and vascular function. In the current study, increased cerebrospinal fluid levels of eotaxin, IL-2, IL-17, and IL-18 were observed in the placental ischemic rat. Eotaxin (CCL11) is a chemoattractant for eosinophils and basophils. In the central nervous system, eotaxin is released by activated astrocytes and act on microglia to promote glutamate-induced neurotoxicity (Parajuli et al. 2015). Therefore, the increase in CSF eotaxin level in response to placental ischemia suggests potential increased glutamate activity and neurotoxicity. Future studies will determine whether placental ischemia is associated with increased glutamate signaling. A review of the literature revealed no studies reporting changes in eotaxin levels during preeclampsia. However, increased circulating (plasma and serum) levels of IL-2 (Szarka et al. 2010;Molvarec et al. 2011), IL-17 (Mart ınez-Garc ıa et al. 2011Toldi et al. 2011;Darmochwal-Kolarz et al. 2012), and IL-18 (Huang et al. 2005;Seol et al. 2009; El-Kabarity and Naguib 2011) have been shown in preeclamptic patients compared to normotensive pregnant controls. Additionally, infusion of IL-17 into the pregnant rat increases mean arterial pressure and oxidative stress (Dhillion et al. 2012) while reducing IL-17 using the soluble receptor to IL-17 into placental ischemic rats attenuates blood pressure and oxidative stress (Cornelius et al. 2013). Moreover, the lymphatic system has been described as a system for the collection of products from the extracellular space into the cerebrospinal fluid (Iliff and Nedergaard 2013; Mendelsohn *Denotes mean AE SEM of incomplete datasets due to some samples falling below detectable range. Bold text represent statistically significant differences between the means (P < 0.05). The numbers in parentheses represent the number of samples with detectable levels. ND, none detected; NC, not calculated; EGF, epidermal growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; GRO/KC/CINC, Growth-regulated oncogene/keratinocyte chemoattractant/cytokine-induced neutrophil chemoattractant; IP-10, Interferon gamma-induced protein 10; LIX, Lipopolysaccharide-induced CXC chemokine; MCP-1, monocyte chemotactic protein 1; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normal T cell expressed and secreted; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor. and Larrick 2013; Plog et al. 2015). Thus, the increases in cerebrospinal fluid cytokines could indicate increased inflammation at the tissue level, potentially through increased production by neuroglia. Eclamptic patients have been reported to present with various cerebrovascular abnormalities, including cerebral edema. Therefore, this study assessed the brain water content in rats subjected to placental ischemia with or without PTZ treatment. Results indicate that brain water content increased in response to placental ischemia as shown previously (Warrington et al. 2014), but was also increased with PTZ treatment. While there was only a small increase in brain water content in response to placental ischemia and PTZ treatment, the brain sits in the enclosed space of the skull and has limited space to expand. Additionally, this increase in brain water content is consistent with findings from previous studies (Amburgey et al. 2010;Ryan et al. 2011;Warrington et al. 2014Warrington et al. , 2015. Additionally, cerebral edema has been reported in preeclamptic patients (Mitas and Rogulski 2012), eclamptic patients Takeuchi et al. 2005;Hirashima et al. 2006), epileptic patients (Ali et al. 2013), and the placental ischemic model (Ryan et al. 2011;Warrington et al. 2014). Based on the finding that both placental ischemia and PTZ injection increased brain water content, it is possible that seizure activity can induce edema and the presence of edema itself may contribute to increased seizure susceptibility. Neurokinin B (NKB) is a neuropeptide belonging to the family of tachykinins that also includes substance P and neurokinin A. NKB has been shown to be increased in the placenta and circulation of preeclamptic women D'Anna et al. 2004;Liu et al. 2009). NKB is increased in the rat hippocampus following PTZ or kainic acid-induced seizures (Marksteiner et al. 1992) and its receptor, NK3, increases in the cerebellum following kainic acid-induced seizures (R€ oder et al. 1994). In this current study, NKB levels were reduced in the anterior cerebrum of rats subjected to PTZ. This difference in results could be a result of the following: (1) the current study utilized a single injection of PTZ while other studies use kindling methods that require two or more injections of the pro-convulsive substance;

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(2) the current study assessed acute changes (30 min following seizure initiation) while other studies measured changes 24 h to months following seizure initiation. It is very interesting that placental ischemia by itself led to a decrease in NKB expression in the anterior brain, the same region where previous study demonstrated increased edema and bloodbrain barrier permeability (Warrington et al. 2014). It is possible that reductions in NKB expression could be compensatory as NKB has been described as a proconvulsive neuropeptide (Maubach et al. 1998). NKB concentration was significantly increased in the circulation following PTZ injection but placental expression was unchanged. Thus, placental ischemia is not associated with increased production of NKB from the placenta. Because the decrease in NKB expression in the brain was associated with an increase in circulating levels of NKB, it is possible that NKB spillover into the circulation occurred in response to PTZ treatment.
Increased neurokinin B in the circulation could serve to increase blood-brain barrier permeability and contribute to cerebral edema formation. Indeed, neurokinin B has been shown to cause vasorelaxation in cerebral vessels in humans and various animal models (Jansen et al. 1991). Additionally both neurokinin B and its receptor, NK1 have been shown to increase plasma extravasation in the lung and ear (Inoue et al. 1996;Grant et al. 2002a, b). Taken together, PTZ may induce edema through increased circulating NKB levels.
In conclusion, the placental ischemic model presents an excellent animal model for studying mechanisms for increased seizure susceptibility. While there are limitations to this model in that the seizures are not spontaneous but are drug-induced and may not directly mimic what occurs in eclamptic patients, this animal model shares numerous characteristics to the clinical condition of preeclampsia. Thus, by targeting specific pathways altered in preeclampsia, insights can be drawn into specific pathways that may trigger seizure induction and studies can be designed to elucidate whether blocking some of these pathways can improve seizure severity or prevent seizures altogether.


extfieldsofstudy: Biology Medicine

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Protective effect of allicin on high glucose/hypoxia-induced aortic endothelial cells via reduction of oxidative stress

This study was designed to explore the protective effect of allicin on aortic endothelial cell injury induced by high glucose/hypoxia and to investigate the corresponding mechanisms. The primary-cultured murine aortic endothelial cells were subcultured. The third passage of cells was adopted and randomly divided into five groups: The normal group (NG), the mannitol group (MG), the high-glucose/hypoxia group (HG), the allicin group (AG) and the protein kinase C (PKC) inhibitor group (GG). The general morphology was observed under an inverted phase-contrast microscope and cell viability was assessed using the MTT assay. Intracellular reactive oxygen species (ROS) levels in the endothelial cells were quantified using dihydroethidium staining. The levels of 8-hydroxydeoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), NADPH oxidase 4 (Nox4) and hypoxia-inducible factor-1α (HIF-1α) and the activity of PKC were measured using ELISA. A quantitative polymerase chain reaction (qPCR) was adopted to evaluate the mRNA expression of Nox4, HIF-1α and NF-κB. The altered cell morphology observed in HG was notably ameliorated in the AG and GG. The protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC in the HG were higher than those in the other groups. Furthermore, the cell viability in the AG was significantly increased and the protein levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC were significantly decreased compared with those in the HG. The ROS production was found to be increased in the HG cells, while there was a significant decrease in the AG cells. These data indicate that allicin exerts a protective effect against high glucose/hypoxia-induced injury in aortic endothelial cells through its antioxidative action, which may involve the inhibition of the PKC pathway and regulation of HIF-1α.

Introduction
Diabetes mellitus is a serious social health problem, with diabetes-related vascular complications representing a major public health burden. According to statistics, >40% of patients hospitalized with acute myocardial infarction have clinical diabetes, and another 35% exhibit impaired glucose tolerance (1). Excessive production of reactive oxygen species (ROS) and the subsequent increase in oxidative stress under high-glucose conditions play a critical role in this pathology (2). The overproduction of ROS, including superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, can prominently damage nucleic acids, lipids and proteins and result in cellular necrosis, tissue structural damage and functional disorder (3). In the setting of hypoxia, hypoxia-inducible factor-1α (HIF-1α) increases in response to ischemia. HIF-1α is a key regulator of oxygen homeostasis, which restores blood flow to ischemic regions (4); however, sustained and prolonged activation of the HIF-1α pathway induces cell death due to the subsequent activation of p53 and other associated genes (5).
Among the currently available medications for diabetes, natural products have drawn increasing attention. As a traditional Chinese herbal medicine, garlic has been used to treat a range of diseases for thousands of years. Allicin is one of the main active components in garlic and exerts several therapeutic effects, such as promoting insulin sensitivity (6,7), decreasing blood glucose levels (8,9), regulating lipid metabolism (10,11), reducing homocysteine levels (12), attenuating superoxide production (13) and limiting inflammation and fibrogenesis. Given the role of oxidative stress within the etiology and pathogenesis of diabetic complications, the present study utilized an in vitro model of diabetes-associated oxidative stress using aortic endothelial cells cultured under high-glucose/hypoxic conditions to clarify the possible mechanism of allicin on diabetic macrovascular complications.
Cell cultures. Murine aortic endothelial cells of the third passage were used in this study. Cells were grown in ECM at 37˚C with 5% CO 2 and 95% air. The medium was changed every 2 days, and the cells were checked every day using an inverted phase-contrast microscope (CKX41; Olympus Corp., Tokyo, Japan). When the cells had predominantly mixed together and reached ~80% confluence, the medium was abandoned and the cells were gently washed twice with phosphate-buffered saline (PBS), prior to detachment from the culture flask, with the aid of 0.2% (w/v) trypsin for 2-3 min. A single cell suspension was generated, and the cells were plated in 6-, 12-, 24-or 96-well plates.
Grouping and treatments. Allicin injection, X/XO and GF109203x were prepared in serum-free medium at 10 µg/ml, 1 mmol/l/20 U/l and 10 µmol/l, respectively. The murine aortic endothelial cells in the exponential growth phase were randomly divided into five groups: The normal group (NG), the mannitol group (MG), the high-glucose/hypoxia group (HG), the allicin group (AG) and the PKC inhibitor (GF109203x) group (GG). The cells of the NG and MG were respectively incubated with serum-free ECM and mannitol (25 mmol/l) for 24 h. The cells of the HG, AG and GG were respectively incubated with X/XO (1 mmol/l/20 U/l), allicin (10 µg/ml) plus X/XO (1 mmol/l/20 U/l), and GF109203x (10 µmol/l) plus X/XO (1 mmol/l/20 U/l) for 40 min, prior to the supernatant solution being abandoned and the cells being treated with glucose (25 mmol/l), allicin (10 µg/ml) plus glucose (25 mmol/l), and GF109203x (10 µmol/l) plus glucose (25 mmol/l), respectively, for 24 h. Following each of the above treatment regimens, the supernatant and culture cells were collected. The collected cells were resuspended in PBS. In order to measure the components within cells, the freeze-thawing method was adopted to break up the cells.
Cell viability assay. Following the different treatments, the morphological changes were observed under a phase-contrast microscope (Olympus Corp.). In addition, MTT assays were performed to assess the cell viability. First, cell viability was assessed using MTT when the cells were cultured in different allicin concentrations of 50, 20, 10, 5 and 2.5 µg/ml (all diluted in serum-free ECM). In brief, the cells of the six groups (the normal control group and the five groups of different allicin concentrations) were seeded into 96-well plates at a density of 1x10 4 cells per well. Upon reaching ~80% confluence, the cells were incubated with different concentrations of allicin for 24 h. The supernatant was then abandoned, and 200 µl serum-free medium, as well as 20 µl MTT dye solution (5 mg/ml), was added to each well. Following the incubation of the samples at 37˚C for 4 h, the MTT/medium solution was removed and 150 µl dimethylsulfoxide was added to dissolve the formazan product in each well on a concentrating table for 15 min. The optical density of each well was measured using an ELISA microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 290 nm. In addition, the cell viability in the NG, MG, HG, AG and GG was assessed using MTT following treatment with the different interventions for 24 h.
Dihydroethidium (DHE) staining to measure ROS. DHE, which is used as a relatively specific measurement for the superoxide anion, is an oxidative fluorescent dye that undergoes a two-electron oxidation to form the DNA-binding fluorophore ethidium bromide. The DHE (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) staining for superoxide was carried out as previously described (14). Briefly, the cells were treated with the aforementioned interventions for 24 h. Following the removal of the supernatant solution, the cells were cultured in DHE (10 µmol/l) diluted with PBS in a light-protected, humidified chamber at 37˚C for 30 min. Once the cells had been washed twice with PBS to remove the uncombined fluorescence probe, fluorescent images were obtained using a fluorescence microscope. The mean fluorescence intensity was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) for quantification. The generation of superoxide was demonstrated by red fluorescent labeling. Non-stained cells were used as a background control. The average of three DHE-stained images was taken as the value for each group.
ELISA. The levels of 8-OHdG, NF-κB, Nox4, HIF-1α and PKC in the cells were determined by ELISA, according to instructions of each assay kit. Aortic endothelial cells were treated as previously described for 24 h. Following the removal of the supernatant solution, the cells were washed twice with ice-cold PBS, scraped from the plate with trypsin and centrifuged at 1,301 rpm for 5 min at 4˚C. The cells were resuspended in PBS. The freeze-thawing method was performed to break up the cells.
Statistical analysis. Data are presented as the mean ± standard deviation. Analysis of variance was used to compare the mean values of more than two groups. Differences were considered significant at P<0.05. All statistical calculations were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA).

Results
General morphological observation under the inverted phase-contrast microscope. Under high-glucose/hypoxic conditions for 24 h, the aortic endothelial cells became shrunken, the intercellular connection was lessened, some of the cells became exfoliated and a few of the cells were found to be floating in the supernate (Fig. 1, HG). The morphology of the MG cells was not obviously changed compared with that of the NG cells (Fig. 1, MG). Compared with the HG cells, the cells in the AG had more complete cell bodies with visible processes, indicating that allicin played a protective role in the injured cells (Fig. 1, AG).
Viability of aortic endothelial cells. MTT assay revealed no significant differences in cell viability between the normal control group and the 10, 5 and 2.5 µg/ml allicin groups (Fig. 2, A3, A4 and A5); however, the cell viability of the 50 and 20 µg/ml allicin groups was significantly decreased compared with that of the normal control group (P<0.01) (Fig. 2, A1 and A2). Based on these findings, 10 µg/ml allicin was selected as the final treatment concentration. In addition, as shown in Fig. 3, an evident decrease in viability was observed in the cells under high-glucose/hypoxic conditions compared with the cells of the NG (P<0.01); however, in experimental  Effect on ROS. ROS production in the endothelial cells of the five groups was assessed by DHE staining. DHE is a fluorescent dye that specifically reacts with intracellular ROS and is converted to the red fluorescent compound ethidium, which then binds irreversibly to double-stranded DNA and appears as nuclear staining. As shown in Fig. 4, the intensity of DHE fluorescence in the endothelial cells in the HG was significantly enhanced compared with that in the NG; allicin significantly downregulated the levels of ROS (P<0.01).
Protein levels of 8-OHdG, NF-κB, Nox4 and HIF-1α and activity of PKC. In the HG, the protein levels of 8-OHdG, NF-κB, Nox4 and HIF-1α and the activity of PKC were significantly increased compared with those of the NG (all P<0.01). In the AG, however, the five parameters were notably restored, showing a significant decrease compared with the levels in the HG (P<0.05 or P<0.01) (Fig. 5).   In the HG, the NF-κB, Nox4 and HIF-1α mRNA expression was found to be significantly increased compared with the expression in the NG (P<0.01), which suggested that high glucose/hypoxia could induce the expression of these mRNAs (Fig. 6). Compared with the HG, however, the mRNA expression of NF-κB, Nox4 and HIF-1α in the AG was significantly decreased (P<0.05 or P<0.01).

Discussion
In diabetes mellitus, the risk of atherosclerosis is enhanced, which results in an increase in the incidence of both cardiovascular and cerebrovascular diseases, such as myocardial infarction and cerebral ischemic attacks. Cardiovascular disease is the major cause of morbidity and mortality in diabetic patients (16). The exact mechanisms responsible for this accelerated development of atherosclerosis in diabetes have remained elusive, but oxidative stress appears to play a major role. Several studies have reported that the overproduction of ROS by the mitochondrial electron transport chain is responsible for hyperglycemia-induced oxidative damage and the pathogenesis of diabetic complications (17)(18)(19)(20)(21).
Hyperglycemia promotes glucose oxidation and protein glycation, impairs DNA repair, with resultant DNA cleavage, and generates ROS, thereby leading to increased oxidative stress (22). Oxidative stress is considered to be the final common pathway through which the hyperglycemia-related pathways (PKC and polyol) can trigger the chronic complications of diabetes. Cumulative oxidant-mediated damage and cellular dysfunction are a result of an imbalance between ROS generation and antioxidant capacity. Furthermore, studies have found that there is a hypoxic microenvironment in diabetes, which is closely associated with the oxidative stress induced by hyperglycemia and plays an important role in diabetic complications (Amandine,Chavez). The development of therapeutic strategies aimed at the removal of free radicals or the prevention of their formation is therefore necessary.
To determine the injury elicited in aortic endothelial cells by high glucose/hypoxia and investigate whether allicin would effectively ameliorate the damage, the expression of oxidative stress-related markers was examined in endothelial cells cultured in high glucose (25 mmol/l for 24 h) and hypoxia [X/XO (1 mmol/l/20 U/l) for 40 min], simulating a diabetic microenvironment in vivo. The possible associated mechanisms were also explored. Since GF109203x is a highly specific PKC inhibitor, it was selected for use as a control. The study showed that the cells in the HG became shrunken and intercellular connections were lessened; furthermore, some of the cells became exfoliated and a few of the cells were observed to be floating. The MG was established to counteract the possible cellular injury by hyperosmosis, and it was found that the cell morphology of the MG was not obviously changed compared with that of the NG. Compared with the HG, the cells in the AG had more complete cell bodies with visible processes, which indicated that allicin played a protective role in the injured cells. Consistent with the MTT results, it was observed that allicin significantly inhibited cell death. Furthermore, a relatively abundant generation of ROS was measured in the cells of the HG by the DHE fluorescence probe. As shown in Fig. 4, allicin significantly downregulated the levels of ROS, showing its effective attenuation of the oxidative stress. The increase in ROS production observed in the endothelial cells of the HG was associated with an increase in the generation of Nox4 and 8-OHdG. NADPH oxidases of the Nox family are major sources of ROS, and the level of 8-OHdG is considered to be a marker of oxidative DNA damage (23). Our preliminary results confirmed the enhancement of oxidative damage to DNA in the HG; however, the damage was markedly attenuated when the cells were treated with allicin. These data suggested that allicin had significantly protective antioxidative effects against high glucose/hypoxia-induced injury in aortic endothelial cells.
To the best of our knowledge, the persistent upregulation of PKC is recognized as an initial event leading to insulin resistance, cardiac disease and nephropathy in diabetes. Numerous studies (24)(25)(26) have previously reported that multiple PKC isoforms are activated in the vascular tissue of diabetic animal models, and the overactivation of the PKC pathway is a key mediator of diabetic vascular complications. The understanding of how hyperglycemia-induced oxidative stress ultimately leads to tissue damage has been advanced considerably (27), and strategies to reduce oxidative stress may A B C exert favorable effects on the progression of diabetes; however, effective therapy to prevent or delay the development of this damage remains limited (28). The present results revealed that the level of PKC protein significantly increased in the murine aortic endothelial cells under high-glucose/hypoxia conditions, and the increase was mitigated by allicin, which demonstrated that the protective effects of allicin against the injury induced by high glucose/hypoxia involved the inhibition of the PKC pathway. PKC has been noted to contribute to the activation of NADPH oxidases in multiple cells (29). The Nox system is considered to be a key contributor to the generation of ROS in numerous cell types and tissues (30). Increased generation of ROS and impaired antioxidant defenses contribute to oxidative stress in diabetes (21). Previous studies have shown increased levels of NADPH oxidase subunits in the vasculature and kidney tissue of diabetic rodents (31,32). Nox4 was initially identified as a kidney NADPH oxidase, but it has since been shown that Nox4 is also abundant in vascular cells (33). There is additionally evidence that Nox4 contributes to oxidative stress in cardiovascular diseases (34,35). The present study showed a significant increase in Nox4 levels in the HG compared with those in the NG. Allicin significantly reduced the Nox4 mRNA and protein expression. These results suggest that high glucose/hypoxia increased the Nox4 levels, which caused an imbalance between the production of free radicals and the antioxidant defense system. Collectively, these data indicate that high-glucose conditions induce increased oxidative stress by activating the PKC pathway, which in turn mediates the increase in NADPH oxidase activity and Nox4 upregulation. Allicin can inhibit the PKC pathway, decrease Nox4-derived ROS and the endothelial cell injury induced by oxidative stress. The effects of allicin in the present study were similar to those of the PKC inhibitor GF109203x.
NF-κB is a pleiotropic, oxidant-sensitive transcription factor that is required for the transcription of the majority of proinflammatory molecules, including adhesion molecules, enzymes, cytokines and chemokines, which mediate the recruitment and retention of monocytes in the subendothelial space; a key early step in the atherosclerotic process. It has also been indicated that the activation of PKC is involved in the hyperglycemia-induced sustained activation of the transcription factor NF-κB (36). In the present study, as shown in Figs. 4 and 5, a significant increase was triggered by high glucose/hypoxia not only in the NF-κB mRNA level, but also in the NF-κB protein expression. Based on these findings, it is indicated that high-glucose conditions induced the activation of the transcription factor NF-κB through the upregulation of PKC. The upregulation of the PKC pathway leads to the concurrent upregulation of NADPH oxidase and increase in ROS production, which can damage endothelial cells and induce the expression of NF-κB, mediating the recruitment and retention of numerous inflammatory factors that further aggravate oxidative stress. Allicin can downregulate both the PKC activation and NF-κB expression. The inhibition of the PKC pathway should be considered as a target for the treatment of diabetes and its associated complications, and the present study showed that allicin inhibited the PKC pathway with a similar efficacy to that of the PKC inhibitor GF109203x.
Hypoxia has a prominent effect on all diabetic complications (37). HIF-1α is highly labile under normal oxygen conditions; however, under hypoxia it is strongly stabilized by ROS, preventing its hydroxylation and proteasomal degradation (38,39). In the present study, X/XO was used to establish the oxygen radical production system, simulating hypoxic conditions. HIF-1-dependent gene regulation leads to an adaptation to the hypoxic state, allowing the cell to survive. Anoxia, however, can induce the overexpression of HIF-1α, which leads to apoptosis-related gene expression, resulting in cell injury. In the present study, it was found that, dependent on the extent of the high glucose and hypoxia, HIF-1α levels also significantly increased. These results showed that endothelial cells incubated under high-glucose/hypoxic conditions expressed significantly high levels of HIF-1α, which were a risk factor for vascular lesion. In addition, allicin downregulated the expression of HIF-1α.
A broad range of antioxidant properties have been attributed to allicin, including direct scavenging of free radicals, maintenance of glutathione and the endogenous antioxidant redox balance, as well as the enhanced expression of the antioxidant enzymes glutathione peroxidase, glutathione reductase, SOD and CAT (13). Furthermore, allicin can suppress the production of certain inflammatory cytokines that have been implicated in the pathogenesis of diabetic complications, including tumor necrosis factor-α and transforming growth factor-1 (40). Specifically, a decrease in oxidative stress has been associated with reduced diabetes-associated atherosclerotic plaque development. As oxidative stress is associated with diabetic complications, antioxidation may be a key factor in the treatment of these diseases. The results of the present study indicate that allicin exerted beneficial effects on the treatment of macroangiopathy and could inhibit endothelial cell apoptosis and promote cellular survival under high-glucose/hypoxic conditions. These data offer a plausible explanation for the effects of allicin in vivo, which appear to be mediated at least partially by the inhibition of the PKC pathway and the consequent decrease in ROS production. In conclusion, the results of the present study suggest that allicin exerts protective effects via the inhibition of the high-glucose/hypoxia-induced upregulation of the PKC pathway and the subsequent activation of NADPH oxidase, increased ROS production, endothelial cell injury, 8-OHdG release and NF-κB upregulation. Inhibition of the PKC pathway may be the common protective mechanism of allicin in diabetes.


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Ccr2 deletion dissociates cavity size and tau pathology after mild traumatic brain injury

Background Millions of people experience traumatic brain injury (TBI) as a result of falls, car accidents, sports injury, and blast. TBI has been associated with the development of neurodegenerative conditions such as Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). In the initial hours and days, the pathology of TBI comprises neuronal injury, breakdown of the blood–brain barrier, and inflammation. At the cellular level, the inflammatory reaction consists of responses by brain-resident microglia, astrocytes, and vascular elements as well as infiltration of peripheral cells. After TBI, signaling by chemokine (C-C motif) ligand 2 (CCL2) to the chemokine (C-C motif) receptor 2 (CCR2) is a key regulator of brain infiltration by monocytes. Methods We utilized mice with one or both copies of Ccr2 disrupted by red fluorescent protein (RFP, Ccr2RFP/+ and Ccr2RFP/RFP). We subjected these mice to the mild lateral fluid percussion model of TBI and examined several pathological outcomes 3 days later in order to determine the effects of altered monocyte entry into the brain. Results Ccr2 deletion reduced monocyte infiltration, diminished lesion cavity volume, and lessened axonal damage after mild TBI, but the microglial reaction to the lesion was not affected. We further examined phosphorylation of the microtubule-associated protein tau, which aggregates in brains of people with TBI, AD, and CTE. Surprisingly, Ccr2 deletion was associated with increased tau mislocalization to the cell body in the cortex and hippocampus by tissue staining and increased levels of phosphorylated tau in the hippocampus by Western blot. Conclusions Disruption of CCR2 enhanced tau pathology and reduced cavity volume in the context of TBI. The data reveal a complex role for CCR2+ monocytes in TBI, as monitored by cavity volume, axonal damage, and tau phosphorylation.

Background
Traumatic brain injury (TBI) is a common condition in today's society. It is variable in extent and circumstance and imposes enormous suffering and expense. The injury can be sustained in a single event or in a repetitive fashion, can be either closed head or penetrating, and ranges in severity from mild to severe. About 75 % of TBI is closed head in nature and mild in intensity [1]. Further contributing to patient suffering and impact on society, TBI is linked to the development of neurodegenerative conditions. For example, TBI of varying intensity is associated with increased risk or earlier onset of Alzheimer's disease (AD), and repetitive mild TBI is associated with chronic traumatic encephalopathy (CTE) [2][3][4][5][6][7][8].
One common pathological feature of TBI, AD, and CTE is the hyperphosphorylation and mislocalization of the microtubule-associated protein tau (MAPT, here termed tau) [9]. Tau contains numerous potential phosphorylation sites and is a target of multiple kinases. In physiological conditions, tau is phosphorylated at a few sites and is located in axons where it binds to microtubules to stabilize the axonal cytoskeleton. However, in pathological conditions, tau can be phosphorylated at multiple additional sites. The (hyper)phosphorylated tau (pTau) dissociates from microtubules in the axon, translocates to the cell body and proximal dendrites, and aggregates in structures termed neurofibrillary tangles, leading to impaired axonal function [9]. While the mechanisms driving tau phosphorylation and tangle formation are not well established, there is substantial evidence suggesting that inflammation can promote this process [10][11][12].
A prominent feature of TBI is the development of an inflammatory reaction within minutes of the injury event [13][14][15][16][17]. Cells at the site of injury secrete cytokines and chemokines that lead to the recruitment of peripheral immune cells-initially neutrophils, which are quickly replaced by monocytes. Concurrently, there is activation of brain-resident astrocytes and microglia. The inflammatory reaction could contribute to the progression of axonal pathology and tissue damage and is thus a potential target to ameliorate TBI pathology [13,17,18].
Although inflammation increases tau phosphorylation and pTau is detected in central nervous system (CNS) tissues of TBI patients and in animal models, whether post-injury inflammation affects pTau levels in the context of TBI has not been addressed. Here, we used the lateral fluid percussion injury (LFPI) model of TBI [19,20] to study how modulation of the monocytic reaction after TBI influences tau phosphorylation. In the context of TBI, the monocytic population is primarily represented by CD45 lo CX3CR1 hi CCR2 − microglia, CD45 hi CX3CR1 lo CCR2 hi inflammatory monocytes, and CD45 hi CX3CR1 hi CCr2 lo patrolling monocytes [21][22][23][24], but perivascular and meningeal macrophages may also play a role. We employed mice deficient for either chemokine (C-X3-C motif) receptor 1 (Cx3cr1 GFP/GFP mice) or chemokine (C-C motif) receptor 2 (Ccr2 RFP/RFP mice). Ccr2 RFP/RFP mice display reduced infiltration of Ly6C hi inflammatory monocytes into the brain after TBI [25]. Here, Cx3cr1 GFP/GFP mice showed no difference in TBI pathology compared to wild type (WT) mice, but Ccr2 RFP/RFP mice had reduced lesion volume and axonal pathology. Surprisingly, Ccr2 RFP/RFP mice also exhibited increased levels and mislocalization of pTau in the cortex and hippocampus, suggesting that monocyte-dependent inflammation exerts distinct effects on tissue loss as compared to tau phosphorylation after TBI.

Animals and TBI induction
All procedures performed on animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic. We employed two mouse strains (Cx3cr1 GFP/GFP and Ccr2 RFP/RFP ) that allowed us to manipulate the cellular reaction after TBI and at the same time visualize the inflammatory cells of interest [26]. Cx3cr1 GFP/GFP mice (green fluorescent protein (GFP) expression in microglia and patrolling CCR2 lo monocytes) were maintained on the Ccr2 RFP/+ background in which only one copy of Ccr2 is disrupted (red fluorescent protein (RFP) expression in inflammatory CCR2 hi monocytes, some T cells). Similarly, for some experiments Ccr2 RFP/RFP mice were maintained on the Cx3cr1 GFP/+ background. There were no statistical differences in the responses to TBI by Ccr2 RFP/RFP ; Cx3cr1 +/+ and Ccr2 RFP/RFP ; Cx3cr1 GFP/+ mice (data not shown) and the two genotypes were pooled together for analysis. Microglia were identified by Iba1 staining in Ccr2 RFP/RFP mice at 3 days post injury (dpi), before other cell types have upregulated Iba1 expression [27].
To induce TBI, we performed lateral fluid percussion injury as described before [19]. Briefly, 8-10-week-old male and female mice were anesthetized with 100 mg/kg ketamine/10 mg/kg xylazine; the fur on top of the head was shaved, and the skin was cut and moved to the side. A craniotomy with~3 mm diameter was opened on the right side of the central suture, halfway between Bregma and Lambda, without disturbing the underlying dura mater. A modified Leur-Lok hub was placed around the craniotomy and sealed in place with dental acrylic. The mice were allowed to recover from anesthesia and returned to their home cages. On the next day, the mice were anesthetized again and attached to a fluid percussion device (AmScien Instruments FP-302) by the Leur-Lok hub. The device was calibrated to deliver mild injury with pressure intensities between 0.4 and 0.6 atm. After injury, the hub was removed, the skin was sutured, and mice were returned to their home cages to recover. For all experiments, the mice were euthanized 3 dpi.

Tissue staining
Tissue staining was used to evaluate the extent of the inflammatory reaction after TBI, lesion volume, axonal pathology, and tau phosphorylation and localization. At 3 dpi, mice were deeply anesthetized with ketamine/ xylazine and perfused with ice-cold phosphate-buffered saline (PBS) followed by 4 % paraformaldehyde (PFA) in PBS. The brains were isolated and postfixed in 4 % PFA overnight and sectioned on a sliding microtome at 30 μm thickness. During staining, all washes were performed three times for 5 min each in 0.1 % triton X-100 in PBS. Antibody solutions were prepared in PBS unless otherwise noted.
Axonal pathology was assessed with amyloid precursor protein (APP) staining which accumulates in axonal swellings of damaged neurons [28]. The sections were incubated in 0.3 % H 2 O 2 for 30 min at RT to inactivate endogenous peroxidases. Antigen retrieval was performed in 1× Target retrieval solution (Dako Cytomation #S1699) containing 0.5 % Tween in PBS at 95°C for 10-15 min. After blocking in 10 % NGS, primary rabbit anti-APP antibody (Invitrogen #51-2700) was applied overnight at 4°C and secondary biotinylated goat anti-rabbit antibody (Vector BA-1000) was applied for 1 h at RT. The signal was detected using the ABC Elite Kit (Vector PK-6100) and DAB substrate kit (Vector SK-4100) according to the manufacturer's instructions. Sections were allowed to dry and coverslipped with hardset mounting medium (Fisher Scientific #SP15).
For phosphorylated tau, after antigen retrieval and blocking in 5 % NGS and 0.3 % Triton X-100 in PBS for 2 h at RT, the localization of pTau was detected with the mouse anti-AT8 antibody (ThermoSci mn1020, 1:500 dilution in 5 % NGS, 0.3 % Triton X-100 in PBS) overnight at 4°C. The secondary antibody used, goat anti-mouse-Alexa 647 (Invitrogen #A21235, 1:1000 dilution), was chosen not to interfere with the signal of genetically encoded GFP and RFP and was applied for 1 h at RT. The sections were mounted on glass slides and coverslipped with VectaShield with DAPI medium (Vector H-1200).

Imaging and image quantification
Sections stained with GFP (or Iba1) and RFP were imaged on a Leica CTR5500 microscope equipped with a QImaging Retiga EXi FAST 13941 MONO camera at 10× magnification. Tiling of consecutive fields of view was employed to capture the whole section and all sections from a mouse using ImagePro Plus software to drive the microscope. Both GFP/Iba1 (microglia) and RFP (monocyte) channels were recorded. The resulting images were then analyzed with ImageJ software (National Institutes of Health) to calculate (1) lesion cavity volume, (2) the volume of brain tissue (in 4 mm slab) occupied by microglial immunoreactivity, and (3) volume of brain tissue occupied by monocyte immunoreactivity. Initially, each slice was manually outlined; for slices with a TBIinduced cavity, the "whole slice" outline was generated based on where the tissue would be if it were not missing. Then, the cavity, the region around the cavity with increased GFP/Iba1 immunoreactivity, and the region around the cavity with increased RFP immunoreactivity were manually outlined for each brain section. Finally, all sections (~22) for an animal were multiplied by the distance between sections (150 μm) and summed up to obtain the corresponding volumes (cavity, microglia, and monocyte immunoreactivity) as a percentage of the brain volume analyzed (4 mm thickness).
APP-stained sections were also imaged with tiling but in brightfield mode. To quantify axonal damage, the corpus callosum in the ipsilateral side to the injury was manually outlined in ImageJ and the percentage of the corpus callosum with APP immunoreactivity above threshold was calculated. Two sections were stained for each animal and averaged after quantification.
AT8-stained sections were imaged on a fluorescent Leica DM4000 microscope equipped with a QImaging Retiga EXi FAST 1394 MONO camera. Images were acquired at 10-20× magnification with a Cy5 filter cube (band-pass excitation 620/60 nm, band-pass emission 700/75 nm filters, 660 nm dichroic mirror). The same fields were also acquired through a DAPI filter cube (band-pass 360/40 nm excitation, band-pass 470/40 emission, 400 nm dichroic mirror) to localize cell nuclei. Images were taken of the ipsilateral cortex (close to the injury cavity), the underlying ipsilateral hippocampus and the contralateral cortex and hippocampus.
Western blot and pTau quantification pTau levels after TBI were quantified by Western blot. TBI and sham mice were anesthetized with ketamine/ xylazine at 3 dpi and perfused with ice-cold PBS. Naïve mice that did not undergo any surgery were used as a control for the effect of surgery itself; naïve mice were simply anesthetized and perfused. The brains were isolated, the ipsilateral and contralateral hemispheres were separated (right and left for naïve mice), and the cortices and hippocampi were microdissected from each hemisphere. Tissues were homogenized in Tissue Protein Extraction Reagent (Pierce #78150) containing 1 % each of protease and phosphatase inhibitors (Sigma #P8340 and #P5726, respectively).
Images of the developed membranes were acquired on a Li-Cor Odyssey CLx imager.
To quantify the relative levels of pTau across samples, the channels for tau and GAPDH were split and analyzed separately with the ImageJ Gel tool. Both AT8 and Tau5 signals were first normalized to GAPDH to ensure equal sample loading. Then, the relative AT8 signal was normalized to total tau. There were no significant differences in total tau between samples after TBI (data not shown).

Statistical analysis
Data points on the graphs represent individual mice. The average and standard error of the mean are also shown. The effects of genotype and injury intensity were examined by two-way analysis of variance (ANOVA); statistical differences between groups were determined with Tukey's post hoc tests. All significant values in post hoc tests were adjusted for multiple comparisons. Groups were considered to be statistically different if p < 0.05 for both factor effect (in ANOVA) and post hoc tests.

Preventing CCR2 + monocyte infiltration after TBI reduces lesion pathology
The inflammatory reaction is a common feature of TBI. Here, we addressed how modulating monocytic inflammation might affect pathological features of neurodegeneration. We altered the inflammatory reaction in two different ways: by inactivation of Cx3cr1 (in Cx3cr1 GFP/GFP mice), which affects the activation of brain-resident microglia [29] and Ly6C lo patrolling monocytes, or by inactivation of Ccr2 (in Ccr2 RFP/RFP mice), which reduces the infiltration of Ly6C hi inflammatory monocytes and a subset of T cells into the brain after TBI [25]. Intercrossing these two mouse strains allowed us to not only inactivate the genes of interest, but also directly visualize chemokine (C-X3-C motif) receptor 1 (CX3CR1)-and CCR2-positive cells in tissue sections [26]. CX3CR1 + patrolling monocytes and CCR2 + T cells represent only a small proportion of infiltrating cells after TBI compared to CX3CR1 + microglia and CCR2 + inflammatory monocytes, respectively (data not shown). Hence, we herein refer to GFP-positive cells as microglia and RFP-positive cells as infiltrating inflammatory monocytes.
Ccr2 RFP/RFP , Cx3cr1 GFP/GFP , and control doubleheterozygous mice were subjected to mild LFPI, and the inflammatory reaction and cavity pathology were analyzed 3 dpi from brain sections spanning a 4-mm portion of the brain that encompassed the whole lesion area (Fig. 1). As expected, the injury induced microglial morphological transformation (seen as increased GFP/Iba1 signal) and infiltration of hematogenous monocytes (presence of RFP + cells) at the site of damage in control double-heterozygous Cx3cr1 GFP/+ ; Ccr2 RFP/+ mice (Fig. 1a). The increased GFP signal and presence of RFP + cells were mostly restricted to the site of tissue damage and brain cavitation. Moreover, the GFP + cells had mainly ramified, bushy, morphology, suggesting that at this early time point, these cells were likely activated microglia rather than infiltrating CX3CR1 + monocytes. Interestingly, although care was taken not to disturb the dura mater and underlying brain during craniotomy, the "sham" surgery itself induced a low level of microglial activation and monocyte infiltration (Fig. 1a). Consistent with previous studies [23,30], deletion of Ccr2 prevented the infiltration of peripheral CCR2-positive cells into the brain after TBI as evident by the absence of RFP + cell staining at the injury site (Fig. 1b). However, there was still prominent microglial reaction as monitored by GFP or Iba1 staining (Fig. 1b). In contrast, neither microglial activation nor monocyte infiltration in response to TBI appeared altered in Cx3cr1 GFP/GFP mice at 3 dpi (Fig. 1c).
We quantified the inflammatory reaction and the cavity pathology by calculating the percentage of the brain parenchyma (in a 4-mm brain portion around the injury) that was occupied by reactive microglia, infiltrating monocytes or size of tissue cavity (Fig. 2). Both injury and genotype had a significant effect on the cavity volume ( Fig. 2a; two-way ANOVA, p < 0.001 for Injury and p < 0.05 for Genotype) and monocytic reaction ( Fig. 2b; two-way ANOVA, p = 0.0010 for Injury and p < 0.01 for Genotype), but there was no significant change in the microglial reaction under any conditions examined ( Fig. 2c; two-way ANOVA, p = 0.0501 for Injury and p = 0.1116 for Genotype). Consistent with the tissue staining ( Fig. 1), Ccr2 deletion resulted in a significant decrease in cavity volume ( Fig. 2a; two-way ANOVA and Tukey's post hoc test, p < 0.05 compared to heterozygous control) and portion of the brain parenchyma containing monocytes ( Fig. 2b; two-way ANOVA and Tukey's post hoc test, p < 0.05 compared to heterozygous control). Despite this, there was no change in the microglial reaction in Ccr2 RFP/RFP mice ( Fig. 2c; two-way ANOVA and Tukey's post hoc test, p = 0.9606).
Deletion of Cx3cr1 did not affect the inflammatory reaction or cavity volume when comparing knockout to heterozygous mice (Fig. 2a-c; two-way ANOVA and Tukey's post hoc test, p = 0.2205 for cavity volume, p = 0.0781 for monocytic reaction, and p = 0.1463 for microglial reaction). Because Cx3cr1 deletion did not affect either the inflammatory reaction or cavity volume after mild injury here, we continued all subsequent studies only with Ccr2 RFP/RFP mice and appropriate controls.

Ccr2 deletion reduces TBI-induced axonal pathology
After TBI, axonal pathology is characterized by APPpositive axonal swellings and axonal retraction bulbs indicating axotomy at sites remote to the injury [28]. In the present study, we detected increased APP immunoreactivity, especially in the corpus callosum underlying the injury site at 3 dpi (Fig. 3a). Examining the corpus callosum at higher magnification showed that many of the APP-positive structures seemed to align with the direction of axonal tracts (Fig. 3b). Deletion of Ccr2 appeared to reduce APP immunoreactivity after TBI. Quantifying axonal pathology as the percentage of the corpus callosum with APP immunoreactivity confirmed reduced axonal pathology in Ccr2 RFP/RFP mice at 3 dpi compared to controls ( Fig. 3c; two-way ANOVA and Tukey's post hoc test, p < 0.05).

Ccr2 deletion alters tau phosphorylation and localization
Because inflammation has been shown to modulate tau pathology and Ccr2 deletion reduces monocyte-mediated inflammation after TBI [10-12, 23, 30], we examined the effect of TBI on tau phosphorylation in Ccr2 RFP/RFP mice. We initially analyzed changes in tau phosphorylation by Fig. 1 Modulation of the inflammatory reaction at 3 days after LFPI in CCR2 and CX3CR1 transgenic mice. Control Ccr2 RFP/+ ;Cx3cr1 GFP/+ (a), Ccr2 RFP/RFP (b), and Cx3cr1 GFP/GFP (c) mice were subjected to surgery (craniotomy; Sham mice) or mild injury, and brains were collected 3 days later. Serial sections were stained for GFP (a, b (Mild), and c) or Iba1 (b (Surgery)) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Inflammatory cells are mostly restricted to the site of injury. Scale bar on images with whole slices, 1 cm. The brightness on the images was adjusted to allow an easier distinction of the brain sections from the background; all sections in a series were adjusted to the same brightness level. Magnified images show a close up view of microglial and monocyte distribution around the cavity size. Note the reduced number of RFP + cells in Ccr2 RFP/RFP mice. Scale bar for magnified images, 100 μm fluorescent immunohistochemistry to determine the localization of pTau (Fig. 4). Tissue staining for the AT8 pTau epitope showed increased staining at the site of injury in the cortex of both control heterozygous and Ccr2 RFP/RFP mice (Fig. 4a). While the pTau immunoreactivity at the lesion site appeared rather diffuse in heterozygous mice, pTau signal was mostly perinuclear in Ccr2 RFP/RFP mice (Fig. 4c). It should be noted that the physiological location of tau is in the axon; hence, both increased phosphorylation and mislocalization to the cell soma after TBI are abnormal.
Increased tau phosphorylation was also detected remote to the injury at 3 dpi, in the underlying hippocampus in both control and Ccr2 RFP/RFP mice (Fig. 4b).
However, the pattern of pTau immunoreactivity appeared different in the two genotypes. As in the cortex, AT8 immunoreactivity in the hippocampus of heterozygous Ccr2 RFP/+ TBI mice was mostly diffuse and located in the hilus of the dentate gyrus, outside the granule cell layer. In contrast, AT8 signal in the hippocampus of Ccr2 RFP/RFP mice was the highest around cell bodies in the granule cell layer of the dentate gyrus and hilus (Fig. 4b, c). Thus, TBI increases tau phosphorylation in both control and Ccr2 RFP/RFP mice, but only Ccr2 RFP/RFP mice show clear pTau translocation to the cell soma.

Both injury and Ccr2 genotype affect pTau levels by Western blot
In order to quantify the changes in pTau after TBI, we performed Western blot analysis for the AT8 pTau epitope in microdissected cortical or hippocampal lysates. Because the hub-placement surgery itself influenced the inflammatory reaction (Fig. 1) and pTau immunoreactivity (Fig. 4a, compare sham ipsilateral and contralateral staining), we also included treatment-naïve mice that did not undergo surgery but received ketamine/ xylazine anesthesia. Interestingly, there was not an injury-and side-dependent increase in pTau levels in the cortex at 3 dpi (Fig. 5a). Yet, mice with both sham surgery and mild LFPI exhibited higher AT8 pTau levels than naïve mice in a side-independent manner (Fig. 5a). The levels of pTau were also modulated differentially in the hippocampus. While hub-placement surgery did not affect pTau in control mice, they increased pTau in Ccr2 RFP/RFP mice in a "dose"and side-dependent manner. That is, even surgery itself led to higher pTau on the ipsilateral side compared to naïve mice, and the higher "dose"-mild injury-led to an even larger increase in pTau (Fig. 5b).
We quantified pTau levels on the ipsilateral and contralateral sides separately in order to evaluate the effects of injury intensity and genotype (Fig. 6). In the ipsilateral cortex, both injury and genotype had a significant effect on pTau levels ( Fig. 6a; two-way ANOVA, p < 0.01 for Injury and p < 0.001 for Genotype). Overall, Ccr2 deletion resulted in higher pTau levels after experimental manipulation of the mice. There was a significant increase in pTau in Ccr2 RFP/RFP mice in sham (surgery) mice compared to naïve mice (two-way ANOVA and Tukey's post hoc test, p < 0.01) but not an additional Fig. 2 Quantification of cavity volume and inflammatory reaction after TBI. Serial sections were stained for GFP (or Iba1) to visualize microglia or RFP to visualize infiltrating bone marrow-derived monocytes (BMDM). Whole slices, lesion cavity, and area of the slices occupied by monocytes (RFP + cells) or microglia (increased GFP or Iba1 immunoreactivity) were manually outlined to calculate their area; all sections were summed up to calculate volumes. Pathology was quantified as a percentage of the analyzed brain tissue (4 mm total thickness) that contained the lesion cavity (a), infiltrating monocytes (b), or increased microglial staining (c). Statistics: two-way ANOVA and Tukey's post hoc test. Comparisons between groups are shown with horizontal lines; vertical line in figure legend indicates main effect of genotype. *p < 0.05; **p < 0.01; ***p < 0.001 increase after mild injury (Fig. 6a). There was a significant difference between control and Ccr2 RFP/RFP mice only for the sham surgery groups (Fig. 6a; two-way ANOVA and Tukey's post hoc test, p < 0.01). Despite the change in pTau immunoreactivity in tissue staining (Fig. 4), there was not a significant difference after injury in pTau levels in control heterozygous mice (Fig. 6a). Ccr2 RFP/RFP mice were also significantly different from control mice on the contralateral side of the cortex (two-way ANOVA, p < 0.05), but there were no significant inter-group changes (Fig. 6b).
Consistent with tissue staining (Fig. 4b), we detected robust changes in pTau levels in the hippocampus on both the ipsilateral and contralateral sides (Fig. 6c, d), with both injury and genotype having significant effects (two-way ANOVA, p < 0.0001 for Genotype for both the ipsilateral and contralateral sides, p < 0.01 and p < 0.05 for Injury intensity for the ipsilateral and contralateral sides, respectively). There was a significant difference in pTau levels after mild injury between control heterozygous and Ccr2 RFP/RFP mice (two-way ANOVA and Tukey's post hoc test, p < 0.0001 for both sides). As in the cortex, the sham surgery induced an increase in pTau in Ccr2 RFP/RFP mice compared to naïve mice that was not subsequently increased by mild injury (Fig. 6c, d; two-way ANOVA and Tukey's post hoc test, p < 0.0001 and p < 0.01 comparing naïve and mild injured Ccr2 RFP/RFP mice on the ipsilateral and contralateral sides, respectively). Experimental manipulation or surgery did not significantly affect pTau levels in control heterozygous mice. Thus, Ccr2 deletion resulted in increased pTau levels on both the ipsilateral and contralateral sides at 3 days after injury or sham surgery. Fig. 3 Effects of CCR2 signaling on axonal damage after TBI. Axonal pathology was evaluated at 3 dpi in control heterozygous and Ccr2 RFP/RFP mice by APP staining. a The injury induces APP accumulation in axons of the ipsilateral side to the injury, particularly in the corpus callosum. Ccr2 deletion decreases APP accumulation in axons. Scale bar, 500 μm. b Higher magnification images of the indicated regions (boxes) show that APP immunoreactivity aligns with axonal tracks in the corpus callosum (CC). Scale bar, 100 μm. c Quantification of axonal pathology as percent area of the corpus callosum with APP immunoreactivity above threshold. Statistics: two-way ANOVA and Tukey's post hoc test. Comparisons between groups are shown with horizontal lines; vertical line in figure legend indicates main effect of genotype. *p < 0.05; **p < 0.01

Discussion
The inflammation that develops after TBI has the potential to influence subsequent neuronal pathology [14,18]; preventing or mitigating aspects of inflammation could help prevent secondary tissue damage and promote recovery [17]. Because inflammation is also a feature of many neurodegenerative conditions, we wanted to examine if modulating inflammation will also affect aspects of neurodegeneration such as altered Tau phosphorylation. We initially examined two models of altered inflammation: deletion of the chemokine receptor CX3CR1, which primarily affects microglia in the brain, and deletion of the chemokine receptor CCR2, which primarily affects bone marrow-derived monocytes and their extravasation to sites of damage [25,29]. Only reduced monocyte infiltration through Ccr2 deletion reduced cavity volume and axonal pathology (Figs. 1, 2, and 3), which led us to examine its effects on tau phosphorylation. Surprisingly, despite the reduced accumulation of monocytes at 3 dpi, Ccr2 RFP/RFP mice showed increased pTau levels by both tissue staining and Western blot analyses (Figs. 4, 5, and 6). Moreover, pTau was translocated from the axon to the cell body in both the cortex and hippocampus of Ccr2 RFP/RFP mice (Fig. 4). The pathology Fig. 4 Assessment of tau phosphorylation and localization by tissue staining. Sections from control heterozygous and Ccr2 RFP/RFP mice after surgery (Sham) or mild injury were stained with the AT8 pTau antibody. Images were taken from the cortex near the lesion cavity (a) and the underlying hippocampus (b) and the corresponding contralateral side. Injury induces increased pTau immunoreactivity as background neuropil staining in control mice and mislocalization to the cell body in Ccr2 RFP/RFP mice. Scale bar, 100 μm. c Insets show pTau staining (AT8, red) in relation to DAPI-positive nuclei (cyan) for the indicated ipsilateral regions (boxes). Scale bar, 50 μm seen in TBI mice here is reminiscent to the neurofibrillary tangles seen in human tauopathies [31] and in patients with CTE or after a single TBI event [3,32,33].
Modulation of inflammation in Cx3cr1 GFP/GFP and Ccr2 RFP/RFP mice The Cx3cr1 GFP/GFP mice that we employed here are commonly used to study and visualize microglia in the brain [29,34]. Indeed, parenchymal microglia are the predominant cell type expressing CX3CR1-and GFP-in the brain as peripheral CX3CR1-positive cells do not cross the intact blood-brain barrier (BBB) in physiological conditions. However, disruption of the BBB in TBI will allow peripheral CX3CR1 + cells to enter the brain. In a dorsal column crush model of spinal cord injury, CX3CR1 + "patrolling" monocytes mediate axonal pathology [35]. In our studies, the great majority of GFP + cells after TBI had ramified or bushy morphology (Fig. 1), suggesting that they are microglia. Yet, additional studies are necessary to establish the ontology of all GFP + cells after TBI.
Delivery of mild TBI to Cx3cr1 GFP/GFP mice did not significantly affect any of the pathological signs we examined: microglial activation, recruitment of CCR2 + cells, or lesion cavity, which was in contrast to the clear effect of Ccr2 deletion (Figs. 1 and 2). Thus, we focused on CCR2 signaling rather than CX3CR1 signaling in subsequent analyses. It should be noted that microglial CX3CR1 signaling has been shown to affect tau phosphorylation in models of tauopathy. Specifically, deletion of Cx3cr1 or its ligand Cx3cl1 results in increased tau phosphorylation in mice overexpressing human tau or the APPPS1 model of AD [11,36,37]. Hence, it is plausible that Cx3cr1 deletion might lead to changes in tau phosphorylation in our mild TBI model, but this question remains to be addressed.
As with Cx3cr1 GFP/GFP mice, Ccr2 RFP/RFP mice are simplistically used to study responses by inflammatory CD45 hi Ly6C hi monocytes [25,26]. However, subsets of T cells, NK cells, and Ly6C lo monocytes express CCR2 or the RFP reporter in some conditions [26]. Although the frequency of these other CCR2/RFP + cells is significantly lower than the frequency of CCR2 + inflammatory monocytes after TBI [24], we cannot rule out that these cell types contribute to the pathology. Thus, additional studies will be necessary to carefully phenotype the different lymphoid populations that respond to TBI and their role in TBI outcomes.

Tau phosphorylation after TBI
There is ample evidence that phosphorylated, aggregated tau is present in the brain of humans that had suffered from various tauopathies, including CTE, AD, frontotemporal dementia, Pick's disease, and others [31]. In the context of TBI, pTau has been observed in numerous individuals who had experienced traumatic events ranging from repetitive sports-related injuries to isolated head traumas (for examples, see 32,3). Yet, there are only a handful of studies that have examined tau phosphorylation , or mild injured control or Ccr2 RFP/RFP mice were probed for pTau (AT8 antibody, green). GAPDH was used as loading control (red). Mild injury induced a visible increase on the ipsilateral side of the cortex and hippocampus in Ccr2 RFP/RFP mice in animal models of TBI. In two different transgenic tau-overexpressing animals, TBI accelerates tau pathology [38,39]. Using wild type mice, Genis et al. [40] and Iliff et al. [41] show that TBI can cause tau hyperphosphorylation in the absence of mutated transgene expression. However, although Genis et al. [40] suggest that tau phosphorylation is transient, peaking at 4 h, Iliff et al. [41] demonstrate increased levels of pTau in the brain even at 28 days after injury. Similar to our study, they found that increased tau phosphorylation is not restricted to the injured ipsilateral side, but is also detectable to the contralateral side.
Another noteworthy observation from our study is that the surgery that is required to prepare mice for fluid percussion injury is sufficient to increase pTau levels when compared to treatment-naïve mice. Furthermore, certain types of anesthetics quickly increase the levels of pTau in the brain [42][43][44]. In our study, all groups received anesthesia, including the treatment-naïve mice. Thus, the effects of anesthesia alone are not sufficient to explain the increase of pTau after surgery and injury. Yet, these findings suggest that tau phosphorylation may represent an immediate physiological response to brain disturbances or a biomarker of altered brain homeostasis. It is worth investigating if tau becomes phosphorylated in other conditions in which brain function is impaired, such as stroke, epilepsy, etc. Moreover, although pTau is more prone to aggregation [9], it remains to be determined if it serves physiological functions that may be aimed at returning the brain to homeostasis in the acute time scales.
We were not able to detect a significant increase in pTau levels after mild injury compared to the surgery itself. One possibility for this is that the surgery and Ccr2 RFP/RFP genotype synergized to produce a strong pTau signal, and the deletion of Ccr2 itself sensitizes the mice to anesthesia-or surgery-mediated effects on pTau. Then, the delivery of an additional injury with mild intensity may not be sufficient to substantially increase pTau levels (Fig. 6). However, despite the lack of protein increase by Western blot, the injury does induce pTau translocation from the axon to the cell body (Fig. 4). As a result, surgery (or other brain disturbances) and traumatic injury may play distinct, sequential roles to lead to frank tau pathology.

Ccr2 modulation in TBI
Preventing monocyte accumulation in the brain has become a promising therapeutic approach to treat TBI and prevent the development of secondary pathologies. Deletion of Ccl2, a ligand for CCR2, Ccr2 itself or antagonism of CCR2 with small molecules have all been shown to be beneficial after TBI [23,30,[45][46][47]. Importantly, Fig. 6 Quantification of pTau Western blots. The levels of pTau (AT8 antibody) were normalized to the levels of total tau (Tau5 antibody). a, b Ccr2 genotype significantly affects pTau levels in both the ipsilateral (a) and contralateral (b) cortex, but pTau levels are elevated after experimental manipulation (surgery or mild injury) only on the ipsilateral side. c, d pTau levels in the hippocampus are increased on both the ipsilateral (c) and contralateral (d) side in injury-and genotype-dependent manner. As in the cortex, sham surgery itself elevates pTau levels in the hippocampus. Statistics: two-way ANOVA and Tukey's post hoc test. Comparisons between groups are shown with horizontal lines; vertical line in figure legend indicates main effect of genotype. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 interfering with CCR2 signaling results in improved cognitive function for up to 4 weeks after the injury.
Yet, here we show that Ccr2 deletion also leads to increased phosphorylation of tau protein-an event that is commonly associated with loss of physiological function and gain of pathological function by tau in a variety of neurodegenerative diseases [31]. In many of these conditions, tau is found in hyperphosphorylated form in the cell body of neurons, similar to what we observed here after TBI in Ccr2 RFP/RFP mice. This raises the question whether interfering with CCR2 signaling might have unexpected consequences of promoting the development of pathologies.
There are still important questions that need to be addressed about the role of CCR2 signaling in tau pathology after TBI. First, are the effects of Ccr2 deletion on tau phosphorylation long lasting or transient? Second, are other disease-associated epitopes of pTau, such as AT180, PHF-1, or CP13, affected? The work by Iliff et al. [41] suggests that tau phosphorylation could persist for at least 28 days after TBI and affect a variety of pTau epitopes. Finally, how can the absence of a peripheral cell type, which is normally not found in the brain, lead to increased tau phosphorylation? One possible answer to this last question is that the infiltrating monocytes modulate the function of resident brain cells such as neurons, astrocytes, and microglia in the context of TBI; in the absence of infiltrating monocytes, the resident cells may release factors that in turn regulate tau phosphorylation. Indeed, we did not detect a reduction in the brain parenchyma occupied by activated microglia in Ccr2 RFP/RFP mice ( Figs. 1 and 2). Whether these microglia are functionally and molecularly different from microglia in control mice after TBI remains to be determined. Similarly, whether the function of other brainresident cell types is affected by the absence of CCR2 + cells in TBI still needs to be examined.

Conclusions
Here we show an unexpected outcome of inhibiting monocyte infiltration into the brain after TBI in Ccr2 RFP/ RFP mice. Although Ccr2 deletion is protective in terms of lesion volume, axonal pathology, and subsequent behavioral outcomes, it leads to increased phosphorylation and mislocalization of tau protein. The latter has been associated with neurodegenerative diseases and should be carefully considered if strategies aimed at CCR2 signaling continue to be pursued for therapeutic purposes in TBI and other conditions.