Angiotensin Receptor Type 1 Inhibition in Lymphopenia and in Neutropenia After Traumatic Brain Injury In Mice: a Randomized Controlled Study (ATLANTIS)


 Background: Cerebral inflammation with invasion of neutrophils and lymphocytes is an important factor in the process of secondary brain damage expansion after traumatic brain injury (TBI). Depletion of neutrophils in mice has been shown to reduce neurologic impairment after TBI. The intrinsic cerebral renin-angiotensin system is an important mediator of cerebral inflammation, as inhibition of the angiotensin II receptor type 1 (AT1) with candesartan improves neurologic recovery, and reduces secondary brain damage and cerebral neutrophil invasion after TBI. The present study was therefore designed to determine the role of immune cells in AT1 inhibition-mediated neuroprotection after TBI. Methods: In study A we assessed the effect of neutrophil depletion in mice after TBI. In study B we investigated the impact of RAG1 deficiency (RAG1-/-; mice without mature B- and T-lymphocytes) after TBI. In study C we investigated the role of neutrophils in candesartan mediated protection after TBI in wild-type mice with and without neutrophil depletion. In study D we examined the role of lymphocytes in AT1 inhibition mediated neuroprotection after TBI in RAG1-/-.Results: Neutropenic and RAG1-/- mice showed reduced brain damage compared to control groups. In control antibody treated wild type mice AT1 inhibition reduced lesion volumes and inflammation compared to vehicle, while in neutropenic mice, candesartan had no effect. In RAG1-/- mice AT1 inhibition resulted in reduction of brain damage and neuroinflammation compared to vehicle group. Conclusion: The present results demonstrate, that reduction of neutrophils and of lymphocytes as well as AT1 inhibition in wild type and RAG1-/- mice reduce brain damage and inflammation after TBI. However, AT1 inhibition was neuroprotective in RAG1-/- mice, but not in neutropenic mice. Therefore, the results indicate that AT1 inhibition mediated neuroprotection may be exerted by anti-inflammatory effects on neutrophils, with a subsequent reduction of neutrophil invasion.


Background
The most common cause of trauma-related death and severe disability in industrialized countries is traumatic brain injury (TBI) (1). Following the mechanical impact (primary injury), detrimental procedure animals were placed in their individual cages and allowed to recover for 6 hours in an incubator heated to 33°C, at a humidity of 35% (IC8000, Draeger, Lübeck, Germany).

Application of antibodies for neutrophil granulocyte depletion and control antibodies
For the depletion of neutrophils in WT mice (studies A and C) the Ly6G-speci c antibody (clone 1A8) was used. In the control antibody group, we used the isotype control antibody immunoglobulin IgG2a (clone: 2A3). Both antibodies, anti-Ly6G (1A8) and IgG2a (2A3) (BXCell; West Lebanon, USA) were diluted in PBS with a nal concentration of 2.5 mg / mL. We injected 0.2 mL (0.5 mg) of anti-Ly6G antibody (ND) and the same volume of the control IgG2a antibody (Ctrl) intraperitoneally (i.p.) 24 h before (studies A and C) and 24 h after experimental TBI (study C).

Application of the AT1 inhibitor candesartan or vehicle solution
In both studies (C, D) candesartan, a speci c AT1 inhibitor was applied. Candesartan (CV-11974; Tocris bioscience; Bristol, UK) and the vehicle solution were prepared and applied as previously described (8): The crystalline form of the active drug candesartan was dissolved prior each set of experiments in 0.037 M Na 2 CO 3 (vehicle solution) in a concentration of 10 µg / mL. The animals received 0.1 mg/kg candesartan (Cand) or vehicle solution (Veh) by subcutaneous (s.c.) injection 30 min after experimental TBI, followed by a daily injection, 24 and 48 h after TBI.

Study B: Effect of RAG1 de ciency mediated lymphopenia 24 hours and 5 days after TBI
In RAG1-de cient mice (RAG1 −/− ) and their RAG1 +/+ wild type litter mates lesion volume and neurology were assessed 24 h (n = 10 / group) and 5 days (n = 8 / group) after CCI. 2.4.2 Study C: Effect of AT1 inhibition in neutrophil depleted mice 3 days after TBI Mice were randomized to treatment (24 h before, and repeated 24 h after TBI) with either anti-Ly6G (ND) or IgG2a control antibody (Ctrl). They were subjected to CCI and then randomly assigned to additional treatment with candesartan (Cand) or vehicle solution (Veh), performed 30 min after TBI and then repeated daily, 24 and 48 h after TBI. Therefore, the animals were randomly allocated to four treatment groups: Ctrl-Cand, Ctrl-Veh, ND-Cand and ND-Veh (n = 12. / group). After the 72-hours observation period, brains were removed for quanti cation of lesion volume, cytokine expression and activated microglia.
Blood samples were withdrawn for hematological quanti cation of white blood cells (WBC), lymphocytes and neutrophils. For comparison we used naïve (non-operated) WT mice (n = 6).

Measurement of physiological parameters
Before, and after experimental TBI body weight of each mouse was controlled. Blood pressure was measured 5 minutes before and after CCI under general anesthesia at the tail using a modi ed NIBP system (RTBP 2000, Kent Scienti c, Torrington, USA; A/D converter: PCI 9112, Adlink Technology, Taiwan; software: Dasylab 5.0, measX, Germany; Flexpro 6.0, Weisang, Germany) as previously described (18). Additionally, blood pressure values were determined in awake animals daily for 8 days before (training phase) and for 2 days after CCI. Perioperative body temperature was measured by a rectal temperature probe (Physitemp; Clifton, NJ, USA).

Assessment of functional outcome
In studies A, C and D neurological outcome was assessed using the rotarod performance test (Heidolph Instruments GmbH &Co.; Schwabach, Germany) as previously described (20)(21)(22)(23). After a pre-training phase (mice remained on a rotating rod for 20 s at 4 rpm) two days before TBI, the time to fall from the accelerating rod in the 2-min test period was registered. This test assesses coordination and motoric function and was performed 1 day before, 24 and 72 h after CCI. In study B functional outcome was determined by Neurological Severity Score (24). In addition to the rotarod test, in studies C and D, functional outcome was also determined by modi ed neurological severity score (mNSS; modi ed after Tsenter et al., 2008 (24)) 1 day before and 24 and 72 hours after CCI (4). To calculate mNSS, general behavior, alertness, motor ability and balance were rated with 6 different tasks. Each task was scored from 0 (normal) up to 3 (failed task). The mNSS ranges from 0 (healthy) to 16 (severely impaired) points (25) (Table 1). All neurological tests were performed by investigators blinded towards experimental group allocations. The modi ed Neurological Severity Score (mNSS) was designed on the basis of the Neurological Severity Score introduced by Tsenter and colleagues (24). The mNSS focusses on motoric function and behavioral de cits and was performed 1 day before CCI and on posttraumatic day 1 and 3 (day 5 in study C) after experimental TBI.

Flow cytometry and blood cell count
At the end of observation period, in deep anesthesia, EDTA anti-coagulated blood samples were taken from the retro-orbital veins as previously described (26). The differential blood cell count was obtained via ow cytometry with a full automated veterinary analyzer, validated for murine blood analyses (ADVIA 2120i Hematology System; Siemens Healthcare, Erlangen, Germany) by an experimenter blinded to experimental group allocation.

Histologic and immunohistochemical evaluation
According to our previous protocol (4) brains were removed in deep anesthesia. For tissue evaluation, the brains were frozen in powdered dry ice and stored at -20°C. They were then cut in coronal plane with a cryostat (HM 560 Cryo-Star, Thermo Fisher Scienti c, Walldorf, Germany) as previously described in detail (8). The rst slide was de ned according to the rst section corresponding to bregma + 3.14 mm in the Mouse Brain Library (www.mbl.org). Sixteen sections (12 and 20 µm) were collected at 500 µm-intervals, placed on Superfrost+TM slides (Thermo Fisher Scienti c, Germany). In cresyl violet (Merck, Darmstadt, Germany) stained sections (12 µm), the total area of both hemispheres and the injured brain tissue area were determined for each section and animal using a computerized image analysis system (Delta Pix Insight; Maalov, Denmark) by an investigator blind to the group allocation. The total hemispheric brain volumes and the lesion volumes were calculated by following formula: 0.  (4,8). Immunohistochemistry was performed as described before (25). For immunohistochemical staining, cryosections (20 µm) were xed in 4 % paraformaldehyde in phosphate buffered saline (PBS), incubated with blocking solution containing serum (5 % goat serum, 2 % bovine serum albumin, Gibco) and 0.1 % TX-100 (Sigma) in PBS for 1 h at room temperature. Primary antibodies speci c for anti-ionized calcium-binding adapter molecule-1 (Iba-1; rabbit anti-mouse, anti-Iba-1 antibody; Wako Chemicals GmbH, Neuss, Germany) were applied in blocking solution overnight at 4°C. The sections were washed, incubated with secondary biotin-conjugated antibodies (goat anti-rabbit IgG; Merck; Darmstadt, Germany) and processed according to the manufacturer's instructions using Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, USA). Images were taken at ×20 magni cation (Axiovert, Zeiss, Germany). The total number of positive cells were counted at bregma -1.28 mm in a region of interest (ROI) of 0.52 x 0.65 mm² in the cortical tissue adjacent to the lesion by an investigator blind to randomization, using ImageJ software (National Institutes of Health, USA). Activated microglia were identi ed as Iba1-immunolabeled cells with bushyround shaped morphology with phagocytic, amoeboid appearance (27). Results are presented in number of activated Iba-1 positive cells / mm 2 .

Gene expression analysis
Brain tissue samples from the lesion and perilesional area of 500 µm coronal cryostat sections between histologic slice intervals were collected, snap frozen in liquid nitrogen, stored at -80°C. As described previously in detail (4, 28), after tissue sampling, extraction of mRNA and cDNA synthesis qPCR were performed (lysis: Qiazol-reagent, Qiagen, Hilden, Germany Real-time RT PCR kits were used according to the manufacturer´s instructions. All assays were carried out by an investigator blinded to group allocation. Using mouse-speci c primers and probes ( Table 2) and optimized temperature conditions for qPCR, absolute copy numbers of the target genes, tumor necrosis factor α (TNFα), transforming growth factor β (TGFβ), interleukin 1β (IL1β), interleukin 6 (IL6) and inducible nitric oxide synthase (iNOS) were calculated, and were then normalized against the absolute copy numbers of cyclophilin A (PPIA) (4,6,8,29). The reference gene PPIA was chosen as single normalizer (30) based on recent ndings in our housekeeping gene study (29). In order to improve comparability of the mRNA expression data between different treatment groups and to eliminate qPCR kit dependent differences and limitations, qPCR data was normalized with PPIA and then related to normalized naïve target gene expression from naïve tissue samples from the corresponding brain region (31). Therefore, normalized target gene expression values are expressed as % naïve expression (4).

Statistical analysis
All experiments were randomized and performed by investigators blinded toward the treatment groups (computer-based randomization: www.pubmed.de/tools/zufallsgenerator). To determine the required sample size, an a priori power analysis using G * Power was performed, with lesion volume data from previously published studies (32). The a priori power analysis was performed to determine an effect size of 0.7, standard statistical power (1 − β) of 0.95, and a signi cance level (α) of 0.05. Statistical analysis was performed using the GraphPad Prism 8 Statistical Software (GraphPad Software Inc., La Jolla, CA, USA). Data distribution was tested by Shapiro-Wilks test. The comparison of parametric and nonparametric data between two independent groups was done using the Welch-t test and the Wilcoxon rank sum test, respectively. In this multi-arm parallel group randomized trial, for comparison of multiple independent groups, if the Shapiro-Wilk normality test was passed, one-way analysis of variance (oneway ANOVA) with post-hoc Holm-Šidák comparisons test (comparisons between all groups) was employed. In experimental groups where two separate treatment factors (neutrophil depletion and AT1 inhibition) are present, a two-way analysis of variance (two-way ANOVA) was performed. Physiologic data, blood cell count, lesion volumes, number of activated microglia and mRNA expression data were compared between experimental groups with two-way ANOVA and post hoc with all-pairwise multiple comparison procedures (Holm-Šidák method). To evaluate group differences in repeated measurements from the same animals (body weight, systolic blood pressure), repeated measures (RM) two-way ANOVA (two-factor repetition) was applied (factors: treatment and time), followed by Šidák's multiple comparisons test. Whenever there were missing values in the repeated measures dataset and a two-way ANOVA was not possible, repeated measures (mNSS, rotarod) data were analyzed with the mixed effect model using the restricted maximum likelihood (REML) method with Holm Šidák's multiple comparison test. The p values were adjusted for multiple comparisons. Values of p < 0.05 were considered signi cant. Data sets were tested for statistically signi cant outliers using the Grubbs' test. Data are presented as mean and standard deviation (mean ± SD).

Perioperative physiological parameters were stable in all groups.
Peri-and intraoperative body temperature and systolic blood pressure were in all mice within physiological range and there was no considerable difference between groups (Tab. 4). As published earlier, in our standardized anesthesia and operation setting, values were stable and within normal physiological limits (6) 3.2 After TBI bodyweight was not affected by anti-Ly6G, RAG1-de ciency or AT1 inhibition.
In naïve mice bodyweight was 24.7 ± 2.2 g. Before TBI, initial bodyweight was in Ctrl-4h mice 25.6 ± 0.9 g and in ND-4h mice 25.5 ± 1.1 g. In ND-24h mice bodyweight was reduced in both treatment groups, without difference between Ctrl and ND (before TBI: 25 In all groups there is a signi cant posttraumatic decrease of bodyweight with a minimum on day 2 and an increase on day 3. However, neither neutrophil depletion, nor RAG1 de ciency, nor AT1 inhibition affected posttraumatic body weight loss compared to control antibody, wild type or vehicle solution treated mice (Tab. 3). 3.3 Low-dose candesartan treatment did not in uence blood pressure after CCI.
In general, the speci c AT1 antagonist candesartan is clinically used for the treatment of arterial hypertension. Therefore, we determined its in uence on arterial blood pressure. In the present study, low dose (0.1 mg / kg) candesartan was applied, which was shown not to alter blood pressure in previous studies (4,8). During the time after CCI in all experimental treatment groups, blood pressure was within physiological range and was not affected by repeated low-dose candesartan (0.1 mg / kg) treatment (Tab. 4).  However, neutrophil depletion did not affect microglial activation in the rst 24 hours after TBI (Fig. 1B).
3.7 Study C: Effect of Neutrophil granulocyte depletion combined with AT1 inhibition 3.7.1: AT1 inhibition had no effect on neutrophil blood cell count.
To analyze an independent effect of candesartan treatment on neutrophil blood cell count we compared all treatment groups by two-way-ANOVA. While a sustained neutrophil depletion was achieved by anti-Ly6G, AT1 inhibition did not affect neutrophil granulocyte count (Fig. 3A).
3.7.2: AT1 inhibition reduced lesion volume in control antibody treated mice, while it had no effect in neutrophil granulocyte depleted mice.
3.7.3: AT1 inhibition reduced cerebral in ammation in control antibody treated mice, while it had no effect in neutrophil granulocyte depleted mice.
We quanti ed the number of perilesional activated Iba-1 positive cells as a marker for microglia activation 3 days after TBI. Activated microglia was assessed at bregma -1.28 mm in cortical perilesional  Fig. 3D). There was a tendency towards lower IL6 expression in vehicle treated ND-mice compared to vehicle treated Ctrl-mice (p=0.09; Fig. 3D). There was a strong tendency towards lower IL1β expression in candesartan treated Ctrl-mice compared to vehicle treated Ctrl-mice (p=0.06; Fig. 3E). TGFβ was elevated in ND-mice by candesartan as well as in Cand-mice by anti-Ly6G-treatment. Gene expressions of TNFα and iNOS were not affected by any treatment.

3.7.4: Neutrophil depletion and AT1 inhibition had no effect on neurological outcome
Neurological assessment was performed 1 day before, and 24 and 72 hours after TBI using a mNSS and time spent staying on the rotarod. Compared to pre-trauma values, CCI induced a highly signi cant impairment in all experimental groups 24 hours after TBI in mNSS (p<0.001; Fig. 3F). Time spent on the rotarod and mNSS improved over time 3 days after TBI without differences between the treatment groups.
3.8 Study D: Effect of RAG1 de ciency and AT1 inhibition 3.8.1 AT1 inhibition reduced lesion volume in RAG1de cient mice.  (Fig. 4C). However, at this time point in ammatory cytokine expression was not affected signi cantly by AT1 inhibition.

AT1 inhibition did not affect neurological outcome in RAG1-de cient mice
Neurological outcome was assessed by mNSS and rotarod at day 1 and 3 after TBI. There was a signi cant increase of neurological impairment (p<0.001). However neurological de cit was not affected by candesartan treatment (Fig. 4D).

Discussion
The purpose of the present study was to explore the so far unexplained role of the innate and of the adaptive cellular immune response in the context of AT1 inhibition after TBI. This study investigated for the rst time the effect of candesartan treatment in neutrophil depleted as well as in RAG1 −/− mice. The results suggest that one possible neuroprotective mechanism of AT1 inhibition may be mediated by neutrophils. Initially, the roles of neutrophils and lymphocytes in the development of secondary brain damage after TBI were analyzed. To this end, we successfully achieved sustained neutrophil depletion by treatment with the speci c anti-Ly6G antibody. Furthermore, RAG1 de ciency, that results in a lack of mature B-and T-cells, showed signi cant lymphopenia. The present data show, that reduction of immune cells of the innate and of the adaptive immune system, neutropenia and lymphopenia, each independently lead to reduced brain damage after TBI. Furthermore, the present results demonstrate that posttraumatic AT1 inhibition reduces histological brain damage and limits immune response in control antibody treated mice with normal neutrophil count and in lymphopenic RAG1-de cient mice. In neutrophil depleted mice, however, AT1 inhibition had no effect on brain damage and neuroin ammation.

Experimental setting and blood cell count
The aim of the present study was to investigate the neuroprotective mechanisms that is mediated by AT1 inhibition and the effects of neutropenia and lymphopenia. To this end we performed the experiments with our established CCI model (4,6,8,29,33,34). The perioperative setting and the conditions are standardized and physiological values, perioperative temperature and blood pressure showed to be stable (6). For the depletion of neutrophils the novel selective antibody against Ly6G (clone 1A8) was chosen, in order to selectively and signi cantly reduce the number of neutrophils without affecting other cell types (35)(36)(37). In contrast, the widely used less speci c antibody for experimental neutrophil depletion anti-Gr1 (clone RB6-8C5) not only reduces Ly6G-speci c cells (neutrophils, Gr1+/Ly6G+), but also decreases the number of other lines of WBC with Ly6-receptors (dendritic cells and subpopulations of monocytes and CD8 T-lymphocytes) (35,38). Therefore, in the present study a sustained selective neutrophil depletion during the whole observation period after TBI was successfully achieved by repetitive application of the speci c monoclonal antibody anti-Ly6G, with a dosage, based on recent studies (36, 39,40). For the assessment of the effects of lymphopenia in TBI the well-established method with RAG1-de cient mice was chosen (41,42). Activated RAG1 plays a key role in VDJ-recombination. RAG1 −/− have small lymphatic organs without mature B-and T-lymphocytes (43) and in the present study they showed to have a signi cant lymphopenia. The observation times of each study were chosen with respect to the maximum brain tissue in ltration of the two immune cell types. After an early posttraumatic liberation of neutrophils from bone marrow and other reservoirs (44) neutrophils are the dominant blood cell population 24 hours after TBI (45). They in ltrate early after TBI into hippocampal and cortical brain tissue (between 4 and 72 hours) and reach a maximum of parenchymal in ltration at 1 day after TBI (46, 47). Therefore, in study A we applied the observation time lengths of 4 and 24 hours post CCI.
Lymphocytes invade into cerebral tissue from the third day on after injury (6, 46). In study B, therefore, we chose the observation time lengths of 1 and 5 days after TBI. To facilitate comparability of the effect of candesartan between neutropenic and lymphopenic mice, we decided to use the observation time length of 72 hours after TBI in studies C and D, respectively (46). In order to ensure timely accurate hematologic analyses and interpretation and to minimize pre-analytic errors a mouse-species-appropriate practical hematologic instrumentation was performed with consistent collection method and appropriate anticoagulation (EDTA) (48). The retroorbital sinus as site of blood withdrawal corresponds more with central blood samples than other peripheral sites (49). The present blood cell count results are consistent with recent data (48). The quality can be considered adequate, as the platelet counts are at normal murine levels and cell clumping occurred rarely (48). Within the rst hours after TBI, there was a reduction of WBC. Due to trauma-associated neutrophil sequestration, a transient leukopenia is part of the in ammatory reaction after TBI (50). In control antibody (IgG2a) treated mice WBC count normalized one day after TBI. In anti-Ly6G treated mice the initial decrease of WBC was more distinct and sustaining with lasting relatively low WBC to day 3 after TBI. However, at each time point, WBC counts were within physiological range (48). In naïve wild type mice lymphocytes are the predominant leukocytes (70 -80 % of WBC). Neutrophils are the most common granulocytes in naïve mice and generally comprise 20 -30 % of WBC count in mice (48). After TBI neutrophils are the most abundant cell population in circulation and cause increased expression of oxidative enzymes (44). In the present study, in Ctrl mice, as a response to TBI, there was a shift from lymphocyte dominated WBC to a signi cant and sustaining elevation of the neutrophil fraction (44,45). In ND mice, in contrast, instead of a posttraumatic increase of neutrophils, there was neutropenia. From 24 hours on after TBI, the fraction of neutrophils was lower in ND mice than in naïve mice, alongside reduced absolute WBC numbers and a compensatory elevation of lymphocytes.
After TBI monocyte counts decreased in all groups, without affection by the selective anti-Ly6G. RAG1 −/− mice showed to be leukopenic and lymphopenia is compensated by elevated neutrophils. In all groups a short-term elevation of hemoglobin and hematocrit in the rst hours after TBI, due to pre-traumatic fasting, were normalized in the following observation periods. Platelets were within physiological range in all groups at all time points without affection by treatment. AT1 inhibition had no effect on posttraumatic blood cell count in wild type, neutropenic and RAG1 −/− mice. In the present study the selective AT1 blocker candesartan was chosen, that crosses the BBB (16). Previous TBI studies demonstrated that neuroprotection and brain damage reduction were achieved at low doses of candesartan that did not affect blood pressure (8), as a decline in blood pressure immediately after TBI may worsen outcome (51).
Furthermore, clinical studies showed bene cial effects of candesartan, independent of the blood pressure lowering effect (52). Therefore, in the present study low dose candesartan (0.1 mg/kg) was applied that has been shown not to affect blood pressure previously (4,8,12,17). In order to achieve a sustaining AT1 inhibition, treatment was started 30 minutes after TBI and then repeated daily (8). As a result, in the present study, during the whole observation time, posttraumatic blood pressure was not affected by candesartan. The peritraumatic body weight was assessed as a surrogate parameter of well-being and intake of food and water. Neither neutrophil depletion, nor candesartan treatment, had any effect on postoperative body weight loss, that had a maximum on day 2, with recovery on days 3 and 5.

Depletion of neutrophils and lymphopenia lead to reduced brain damage and in ammation
Neutrophils are an abundant proin ammatory population of circulating leukocytes that are usually among the rst responders to tissue injuries in the periphery and the central nervous system (53). After trauma neutrophil sequestration plays a major role in multiple organ failure (50). In the early phase after TBI, neutrophils are the dominating immune cells that in ltrate damaged brain tissue (47), causing an increase of brain damage (46). Evidence suggests that depletion of neutrophils may have bene cial effects in the early phase after TBI. Recent studies have revealed that depletion of neutrophils with anti-Gr-1 antibodies reduced neuroin ammation and brain tissue loss up to seven days after TBI (54). Actually, in the present study, neutrophil depletion reduced brain damage 24 hours after TBI compared to control group. Alongside the damage reduction, there was a reduction of in ammatory TNFα levels.
Although T cells play diverse roles in adaptive immune responses and the regulation of in ammation, their role in TBI pathogenesis is unresolved (55). Our recent data indicated that cerebral T cell in ltration aggravated neuroin ammation but did not increase lesion volume after TBI (56, 57). In a previous closedhead injury study no difference in any pathologic or neurologic parameters was observed between wildtype and RAG1 −/− mice up to 7 days after closed head injury (41). The authors concluded, that adaptive immunity is not of crucial importance for initiating and sustaining in ammatory neuropathology after closed head injury (41). In another TBI (aseptic cerebral injury) study, however, Fee and colleagues demonstrated that CD4 + T lymphocytes contribute to the severity of the acute phase of TBI and that brain injury is attenuated in RAG1 −/− mice compared to wild type animals (42). In accordance with these results, in the present study, RAG1 de ciency lead to reduced lesion volumes and neuroin ammation 1 and 5 days after TBI.
However, 24 hours after TBI, neutrophil depletion showed to be more effective in reduction of lesion volume (33 % compared to non-depleted wild type) than lymphopenia (17 % compared to wild type). It has been shown that acute posttraumatic cerebral in ltration of neutrophils is more distinct, compared to in ltration of lymphocytes (46). Therefore, it is possible that a reduction of neutrophil in ltration may have a stronger anti-in ammatory effect in the rst days after TBI, than a reduction of lymphocyte in ltration. This may explain why in studies with neutrophil depletion brain damage and in ammation are consistently reduced (54) and why in studies with RAG1 de ciency the results are inconsistent (41, 42, 56).
4.3 AT1 inhibition reduced histological brain damage and microglial activation in non-neutrophil depleted mice and RAG1 de cient mice, whereas candesartan treatment failed to be neuroprotective in neutrophil depleted mice Growing amount of evidence shows that the entire cellular immune response (granulocytes, monocytes, lymphocytes, etc.) is modulated by the RAS (58, 59). Angiotensin II, via AT1 is one of the most important inducers and perpetuators of cerebral in ammation and oxidative stress (7,60). AT1 is widely expressed in the mature central nervous system, mainly in neurons, endothelial and smooth muscle cells, astrocytes and microglia, important modulators of neuroin ammation (9,61). AT1 is also expressed on migrating immune cells, like neutrophils, macrophages and T-cells. AT1 activation initiates subsequent production of chemokines, cytokines, and adhesion molecules, which contribute to the immigration of these activated immune cells into the lesion (7,(62)(63)(64)(65). They induce in ammatory responses and release high levels of ROS by activation of NADPH oxidase. AT1 signaling modulates the NADPH oxidase complex activation and induces transcription of several pro-in ammatory cytokines by activation of NF-κB dependent transcription (15,66). This leads to subsequent stimulation of several different kinases that themselves participate in the propagation of in ammatory responses and apoptotic pathways (17,(67)(68)(69)(70).
In accordance with recent studies the present study showed that repeated posttraumatic treatment with the selective AT1 inhibitor candesartan resulted 3 days after TBI in a reduced histological brain damage in mice with normal posttraumatic neutrophil count. AT1 inhibition additionally decreased microglial activation and pro-in ammatory signaling in these mice (8, 12). In the present study AT1 blockade mediated brain damage reduction was accompanied by a decrease of microglial activation, respectively. Activated microglia were reduced by app. 14 % by candesartan and by app. 7 % by neutrophil depletion compared to vehicle and control antibody treatment. Therefore, earlier ndings in candesartan treated mice, where number of neutrophils and activated microglia were reduced 3 days after TBI (4,17) are con rmed in the present study. The pleiotropic cytokine TNFα is produced in microglia, astrocytes and neurons. It is involved in BBB-dysfunction and transmigration of WBC into brain tissue and induces neuronal loss via microglial activation and phagoptosis of neurons (2,(71)(72)(73). It has been demonstrated earlier that microglial release of TNFα is reduced by AT1 antagonists (7,74). After an early upregulation in the rst 8 hours after TBI (72) TNFα decreases signi cantly in the following time (75). This kinetic could explain that in the present study there is only a reduction of TNFα 4 hours after TBI in ND mice. One of the most potent key pro-in ammatory cytokines is IL1β. It plays a major role in leukocyte adhesion, BBB-dysfunction, brain edema and apoptosis as well as in the induction of other pro-in ammatory cytokines. Clinical studies showed a correlation between increased IL1β levels and elevated ICP with worse prognosis (76). Reduced activity of IL1β showed improved neurological outcomes and reduced in ltration of neutrophils (72,(77)(78)(79). Cerebral IL1β expression increases in the rst hour after TBI and reaches highest levels 12 and 24 hours after experimental TBI, and remains elevated up to 48 hours (80).
In the present study there is a strong tendency towards lower IL1β expression in candesartan treated control-antibody mice. However, the lack of effect of RAG1 de ciency or neutropenia on IL1β expression in the present study may be due to low cytokine mRNA levels 3 days after CCI (15). In a recent study, we could demonstrate reduced cytokine expression by AT1 inhibition 12 hours after TBI (8). Several studies have shown that the cytokine IL6 is up-regulated after TBI with consecutive increased microglia activation and neurological impairment (2,81). Clinical studies postulate a correlation between elevated IL6 serum levels, ICP increase and severity of TBI (82). Furthermore, IL6 regulates migration of neutrophils during acute in ammation (83). In the present study IL6 is reduced by AT1 inhibition three days after TBI in control antibody treated mice, whereas in neutropenic mice, candesartan did not affect IL-6 expression.
In a recent study we could demonstrate that posttraumatic AT1 inhibition improved neurological recovery, reduced histological brain damage and limited immune response in young adult and aged mice. We postulated that the protective effect is attributed to a diminished microglia activation and increased antiin ammatory microglia polarization. One major nding was that neutrophil in ltration was largely reduced (4). As AT1 is expressed on circulating neutrophils and lymphocytes (64, 84), several studies indicate that in ltrations of both immune cell types, neutrophils and lymphocytes are reduced by AT1 blockade (85, 86). However, in our recent study perilesional T-cell immigration was not affected by AT1 inhibition (4). Neuroprotective mechanisms of AT1 inhibition in the acute phase after TBI may therefore be independent of adaptive lymphocyte reaction. A recent study showed that expression of CD62L on human neutrophils is modulated by AT1 receptors, on pathways involving extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and calcineurin (87), leading to reduced transmigration of neutrophils. Recent studies revealed that AT1 inhibition leads to down regulation of important recruitment proteins like ICAM1 in endothelial cells and CD11b/CD18 on WBC and the post traumatic increase of BBB permeability. Consecutively, the in ltration of immune cells is largely reduced by AT1 inhibition (85, 86, 88, 89). A recent murine cerebral transcriptomic analysis after TBI showed strong alterations of gene transcription by candesartan treatment. The authors point to a role for candesartan in altering many different aspects of the response to TBI, particularly those involved with cellular response to stress, extracellular matrix alterations and the innate immune response (90). Therefore, AT1 inhibition may have a direct and modulating antiin ammatory effect on invading neutrophils and resident activated microglia (17). The reduction of the cerebral in ammatory response of the innate immune system with reduced microglial activation as well as decreased in ltration of neutrophils may be a putative protective mechanism of AT1 inhibition mediated anti-in ammation and neuroprotection (4).

Conclusions
In ammatory response is believed to play a key role in TBI with detrimental effects on brain damage. In order to prove the hypothesis, that AT1 inhibition may constitute an effective neuroprotective strategy against exacerbated neuroin ammation after TBI by affecting the adaptive and the innate immune system, the present study was performed. For the rst time the effects of AT1 inhibition in neutrophil depleted and in RAG1 −/− mice were investigated. The present data show, that reduction of immune cells of the innate and of the adaptive immune system, neutropenia, by anti-Ly6G treatment, and lymphopenia, in RAG1 −/− mice, each independently lead to reduced brain damage after TBI. Furthermore, the present results demonstrate that posttraumatic AT1 inhibition reduces histological brain damage and limits immune response in control antibody treated mice with normal neutrophil count and in lymphopenic RAG1-de cient mice. AT1 inhibition was highly effective in animals to reduce brain damage by reducing microglial response. In neutrophil depleted mice, however, AT1 blockage had no effect on brain damage and neuroin ammation. We conclude, that protective effects of AT1 inhibition are independent of lymphocytes but dependent on neutrophils. Therefore, AT1 inhibition may exert its neuroprotective properties by affecting neutrophil invasion into injured brain tissue. AT1 inhibition is a promising therapeutic strategy to limit secondary brain damage after TBI. The present data may help to understand the TBI pathophysiology and develop optimal pharmacologic intervention for TBI patients. RTK is the corresponding author. He was the major contributor to study conceptions and acquisition of funding. He planned and designed the studies, performed and supervised the experiments, analyzed and interpreted the data regarding the hematological, histological, molecular biological and neurological assessments. RTK wrote the manuscript. SPCC made substantial contributions in planning and designing the studies, performed the histological and molecular biological examination of the brains, she helped with hematological examinations and prepared the blood cell count. She was a major contributor in writing the manuscript. TK performed experiments, blood withdrawals and histological examinations, he made substantial contributions in data acquisitions. AVH performed experiments and histological examinations, made substantial contributions in data acquisitions. MKS made substantial contributions in data analysis and interpretation regarding molecular biology. AS contributed substantially in data acquisition of histology and molecular biology. SCT was a major contributor in study conception, data interpretation, and writing the manuscript. All authors read and approved the nal manuscript.