Systemic Administration of an Anti-CD18 Antibody Prevents the Accumulation of Peripheral Leukocytes in Pericontusional Tissue, but Does Not Improve Outcome after Traumatic Brain Injury in Mice.

Brain edema formation is a key factor for secondary tissue damage after traumatic brain injury (TBI), however, the type of brain edema and the temporal profile of edema formation are still unclear. We performed free water imaging, a bi-tensor model based diffusion MRI analysis, to characterize vasogenic brain edema (VBE) and cytotoxic edema (CBE) formation up to 7 days after experimental TBI. Male C57/Bl6 mice were subjected to controlled cortical impact (CCI) or sham surgery and investigated by MRI 4h, 1, 2, 3, 5, and 7 days thereafter (n = 8/group). We determined mean diffusivity (MD) and free water (FW) in contusion, pericontusional area, ipsi- and contralateral brain tissue. Free (i.e., non-restricted) water was interpreted as VBE, restricted water as CBE. To verify the results, VBE formation was investigated by in-vivo 2-Photon Microscopy (2-PM) 48h after surgery. We found that MD and FW values decreased for 48h within the contusion, indicating the occurrence of CBE. In pericontusional tissue, MD and FW indices were increased at all time points, suggesting the formation of VBE. This was consistent with our results obtained by 2-PM. Taken together, CBE formation occurs for 48h after trauma and is restricted to the contusion, while VBE forms in pericontusional tissue up to 7 days after TBI. Our results indicate that free water magnetic resonance imaging may represent a promising tool to investigate vasogenic and cytotoxic brain edema in the laboratory and in patients.

The role of the endothelial contractile apparatus in the process of brain edema formation after brain trauma is not characterized. Phosphorylation of myosin light chains by myosin light chain kinases (MLCK) activates endothelial contractile elements and results in a rearrangement of the cytoskeleton. This may enhance post-traumatic blood-brain barrier dysfunction. In order to investigate the role of the MLCK on brain edema formation and blood-brain barrier permeability after brain injury, mice were anesthetized and subjected to a controlled cortical impact (CCI). MLCK expression is significantly up-regulated after CCI with a maximum 12 h post-injury. Specific inhibition of MLCK by ML-7 resulted in a reduction of phosphorylation of myosin light chains and improved blood-brain-barrier integrity. Accordingly, ML-7 attenuated post-traumatic brain edema formation and intracranial hypertension 24 h after CCI. Prevention of brain edema formation did not translate into improved neurological outcome or reduced brain lesion. In conclusion, the results confirm that the endothelial contractile apparatus is activated by CCI and opens the endothelial barrier leading to vasogenic brain edema formation. Lack of neurological and histological improvement suggests that specific targeting of vasogenic brain edema at the endothelial level is not sufficient to limit secondary brain damage and has, therefore, to be combined with other potential neuroprotective strategies.

Many compounds tested for a possible neuroprotective effect after traumatic brain injury (TBI) are not readily soluble and therefore organic solvents need to be used as a vehicle. It is, however, unclear whether these organic solvents have intrinsic pharmacological effects on secondary brain damage and may therefore interfere with experimental results. Thus, the aim of the current study was to evaluate the effect of four widely used organic solvents, dimethylsulfoxide (DMSO), Miglyol 812 (Miglyol®), polyethyleneglycol 40 (PEG 40), and N-2-methyl-pyrrolidone (NMP) on outcome after TBI in mice. A total of 143 male C57Bl/6 mice were subjected to controlled cortical impact (CCI). Contusion volume, brain edema formation, and neurological function were assessed 24 h after TBI. Test substances or saline were injected intraperitoneally (i.p.) 10 min before CCI. DMSO, Miglyol, and PEG 40 had no effect on post-traumatic contusion volume after CCI; NMP, however, significantly reduced contusion volume and brain edema formation at different concentrations. The use of DMSO, Miglyol, and PEG 40 is unproblematic for studies investigating neuroprotective treatment strategies as they do not influence post-traumatic brain damage. NMP seems to have an intrinsic neuroprotective effect that should be considered when using this agent in pharmacological experiments; further, a putative therapeutic effect of NMP needs to be elucidated in future studies.

Vasogenic brain edema (VBE) formation remains an important factor determining the fate of patients with traumatic brain injury (TBI). The spatial and temporal development of VBE, however, remains poorly understood because of the lack of sufficiently sensitive measurement techniques. To close this knowledge gap, we directly visualized the full time course of vascular leakage after TBI by in vivo 2-photon microscopy (2-PM). Male C57BL/6 mice (n = 6/group, 6-8 weeks old) were assigned randomly to sham operation or brain trauma by controlled cortical impact. A cranial window was prepared, and tetramethylrhodamine-dextran (TMRM, MW 40,000 Da) was injected intravenously to visualize blood plasma 4 h, 24 h, 48 h, 72 h, or seven days after surgery or trauma. Three regions with increasing distance to the primary contusion were investigated up to a depth of 300 μm by 2-PM. No TMRM extravasation was detected in sham-operated mice, while already 4 h after TBI vascular leakage was significantly increased (p < 0.05 vs. sham) and reached its maximum at 48 h after injury. Vascular leakage was most pronounced in the vicinity of the contusion. The rate of extravasation showed a biphasic pattern, peaking 4 h and 48-72 h after trauma. Taken together, longitudinal quantification of vascular leakage after TBI in vivo demonstrates that VBE formation after TBI develops in a biphasic manner suggestive of acute and delayed mechanisms. Further studies using the currently developed dynamic in vivo imaging modalities are needed to investigate these mechanisms and potential therapeutic strategies in more detail.

Interleukin-17 (IL-17) is a proinflammatory cytokine primarily secreted in the brain by inflammatory T lymphocytes and glial cells. IL-17+ T-helper (Th17) cells are increased in the ipsilateral hemisphere after experimental traumatic brain injury (TBI), and IL-17 levels are increased in serum and brain tissue. We hypothesized that il17a and related gene expression would be increased in brain tissue after TBI in mice and il17a–/– mice would demonstrate neuroprotection versus wild type. The controlled cortical impact (CCI) model of TBI in adult male C57BL6/J mice was used for all experiments. Data were analyzed by analysis of variance (ANOVA) or repeated-measures two-way ANOVA with the Bonferroni correction. A value of p < 0.05 determined significance. Expression of il17a was significantly reduced in the ipsilateral cortex and hippocampus by day 3 after TBI, and expression remained low at 28 days. There were no differences between il17a–/– and il17a+/+ mice in beam balance, Morris water maze performance, or lesion volume after CCI. Surprisingly, naïve il17a–/– mice performed significantly (p = 0.02) worse than naïve il17a+/+ mice on the probe trial. In conclusion, sustained depression of il17a gene expression was observed in brains after TBI in adult mice. Genetic knockout of IL-17 was not neuroprotective after TBI. IL-17a may be important for memory retention in naïve mice.

Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using anti‐Gr‐1 antibody was compared to that in mice with intact neutrophil count. Brain edema formation was assessed by Evan's blue dye extravasation and analysis of brain water content. Immunohistochemistry was used to assess cell death and microglial activation. Lesion volume was measured 7 and 14 days after CCI. Neutrophil‐depletion did not significantly affect Evan's blue extravasation at 24 or 48 hours after CCI. However, neutrophil‐depleted mice exhibited decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Additionally, neutropenic mice had a decreased number of activated microglia, and brain tissue loss was attenuated in these mice. Furthermore, the number apoptotic cells 24 h after injury was significantly reduced in neutrophil depleted mice. Our results suggest that neutrophils play a role in the edema formation and contribute to the cell death and tissue loss following TBI.

The attenuation of brain edema is a major therapeutic target after traumatic brain injury (TBI). Vasopressin (AVP) is well known to play a major role in the regulation of brain water content and vasoendothelial functions and to be involved in brain edema formation. Therefore, the aim of the current study was to analyze the antiedematous efficacy of a clinically relevant, nonpeptidic AVP V1a and V2 receptor antagonists. C57Bl6 mice were subjected to controlled cortical impact (CCI) and V1a or V2 receptors were inhibited by using the highly selective antagonists SR-49059 or SR-121463A either by systemic (intraperitoneal, IP) or intracerebroventricular (ICV) application. After 24 h, brain edema, intracranial pressure (ICP), and contusion volume were assessed. Systemically applied AVP receptor antagonists could not reduce secondary lesion growth. In contrast, ICV administration of AVP V1a receptor antagonist decreased brain edema formation by 68%, diminished post-traumatic increase of ICP by 46%, and reduced secondary contusion expansion by 43% 24 h after CCI. The ICV inhibition of V2 receptors resulted in significant reduction of post-traumatic brain edema by 41% 24 h after CCI, but failed to show further influence on ICP and lesion growth. Hence, centrally applied vasopressin V1a receptor antagonists may be used to reduce brain edema formation after TBI.

The formation of brain edema and subsequent intracranial hypertension are major predictors of unfavorable outcome following traumatic brain injury (TBI). Previously, we reported that arginine vasopressin (AVP) receptor antagonists reduce post-traumatic and post-ischemic brain edema in mice. The aim of the current study was to investigate further the contribution of arginine vasopressin V1a receptors to TBI-induced secondary brain damage in V1a receptor knock-out mice. V1a receptor knock-out (V1a -/-) and wild-type mice were subjected to controlled cortical impact (CCI), and edema (brain water content measured before and 24 h after CCI), primary and secondary contusion volume (15 min and 24 h after CCI), neurological function (one day before and seven days after CCI), body weight (before and seven days after CCI) and mortality were measured. Twenty-four h after CCI, V1a receptor knock-out mice had significantly less brain water content than wild-type mice (mean±standard error of the mean: 79.8%±0.3 vs. 80.6%±0.2, respectively), and secondary contusion volume was significantly smaller (38.2±1.7 mm(3) vs. 45.1±1.5 mm(3) in wild-type mice). Furthermore, the V1a receptor knock-out mice had less neurological dysfunction (3.2±0.8 vs. 7.0±1.4 in wild-type mice) and weight loss (1.0±1.0% vs. 4.9±1.8% in wild-type mice) seven days after CCI. Our data show that mice lacking V1a receptors have less secondary brain damage following experimental traumatic brain injury. We therefore conclude that V1a receptors may represent a novel drug target for preventing post-traumatic brain edema.

BackgroundIt remains unclear whether neurobehavioral testing adds significant information to histologic assessment of experimental traumatic brain injury (TBI) and if automated gait assessment using the CatWalk XT®, while shown to be effective in in the acute phase, is also effective in the chronic phase after experimental TBI. Therefore, we evaluated the correlation of CatWalk XT® parameters with histologic lesion volume and analyzed their temporal and spatial patterns over four weeks after trauma induction.MethodsC57Bl/6 mice were subjected to controlled cortical impact (CCI). CatWalk XT® analysis was performed one day prior to surgery and together with the histological evaluation of lesion volume on postoperative days one, three, seven, 14 and 28. Temporal and spatial profiles of gait impairment were analyzed and a total of 100 CatWalk XT® parameters were correlated to lesion size.ResultsWhile in the first week after CCI, there was significant impairment of nearly all CatWalk XT® parameters, impairment of paw prints, intensities and dynamic movement parameters resolved thereafter; however, impairment of dynamic single paw parameters persisted up to four weeks. Correlation of the CatWalk XT® parameters with lesion volume was poor at all timepoints.ConclusionAs CatWalk XT® parameters do not correlate with focal lesion size after CCI, gait assessment using the CatWalk XT® might add valuable information to solitary histologic evaluation of the injury site. While all CatWalk XT® parameters can be used for gait assessments in the first week after CCI, dynamic single paw parameters might be more relevant in the chronic phase after experimental TBI.

BackgroundLeukocytes are believed to be involved in delayed cell death following traumatic brain injury (TBI). However, data demonstrating that blood-borne inflammatory cells are present in the injured brain prior to the onset of secondary brain damage have been inconclusive. We therefore investigated both the interaction between leukocytes and the cerebrovascular endothelium using in vivo imaging and the accumulation of leukocytes in the penumbra following experimentally induced TBI.MethodsExperimental TBI was induced in C57/Bl6 mice (n = 42) using the controlled cortical impact (CCI) injury model, and leukocyte-endothelium interactions (LEI) were quantified using both intravital fluorescence microscopy (IVM) of superficial vessels and 2-photon microscopy of cortical vessels for up to 14 h post-CCI. In a separate experimental group, leukocyte accumulation and secondary lesion expansion were analyzed in mice that were sacrificed 15 min, 2, 6, 12, 24, or 48 h after CCI (n = 48). Finally, leukocyte adhesion was blocked with anti-CD18 antibodies, and the effects on LEI and secondary lesion expansion were determined 16 (n = 12) and 24 h (n = 21), respectively, following TBI.ResultsOne hour after TBI leukocytes and leukocyte-platelet aggregates started to roll on the endothelium of pial venules, whereas no significant LEI were observed in pial arterioles or in sham-operated mice. With a delay of >4 h, leukocytes and aggregates did also firmly adhere to the venular endothelium. In deep cortical vessels (250 μm) LEIs were much less pronounced. Transmigration of leukocytes into the brain parenchyma only became significant after the tissue became necrotic. Treatment with anti-CD18 antibodies reduced adhesion by 65%; however, this treatment had no effect on secondary lesion expansion.ConclusionsLEI occurred primarily in pial venules, whereas little or no LEI occurred in arterioles or deep cortical vessels. Inhibiting LEI did not affect secondary lesion expansion. Importantly, the majority of migrating leukocytes entered the injured brain parenchyma only after the tissue became necrotic. Our results therefore suggest that neither intravascular leukocyte adhesion nor the migration of leukocytes into cerebral tissue play a significant role in the development of secondary lesion expansion following TBI.

BackgroundBrain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear.MethodsIn this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data.ResultsNeutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury.ConclusionOur results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice.

Targeting microthrombosis and neuroinflammation with vepoloxamer for therapeutic neuroprotection after traumatic brain injury.

Suppression of miR-193a alleviates neuroinflammation and improves neurological function recovery after traumatic brain injury (TBI) in mice

Traumatic brain injury (TBI) from blast injury is often complicated by hemorrhagic shock (HS) in victims of terrorist attacks. Most studies of HS after experimental TBI have focused on intracranial pressure; few have explored the effect of HS on neuronal death after TBI and none have been done in mice. We hypothesized that neuronal death in CA1 hippocampus would be exacerbated by HS after experimental TBI. C57BL6J male mice were anesthetized with isoflurane, mean arterial blood pressure (MAP) was monitored, and controlled cortical impact (CCI) delivered to the left parietal cortex followed by 1) continued anesthesia (CCI only), or either 60 or 90 min of volume controlled HS. Parallel 60 or 90 min HS only groups were also studied. After HS (± CCI), 6% hetastarch was used targeting MAP ≥50 mmHg during a 30 min Pre-Hospital resuscitation phase. Then, shed blood was re-infused and hetastarch given targeting MAP ≥60 mmHg during a 30 min Definitive Care phase. Neurological injury was evaluated at 24h (fluorojade C) or 7 days (CA1 and CA3 hippocampal neuron counts). HS reduced MAP to 30−40 mmHg in all groups, p<0.05 vs CCI only. Ipsilateral CA1 neuron counts in the 90 min CCI+HS group were reduced at 16.5±14.1 vs 30.8±6.8, 32.3±7.6, 30.6±2.2, 28.1±2.2 neurons/100 Im in CCI only, 60 min HS only, 90 min HS only, and 60 min CCI+HS, respectively, all p<0.05. CA3 neuron counts did not differ between groups. Fluorojade C staining confirmed neurodegeneration in CA1 in the 90 min CCI+HS group. Our data suggest a critical time window for exacerbation of neuronal death by HS after CCI and may have implications for blast injury victims in austere environments where definitive management is delayed.

Ferroptosis plays an important role in traumatic brain injury (TBI). The p53 protein is a major mediator of ferroptosis. However, the role of p53-mediated ferroptosis in TBI has not been studied. Sirtuin 2 (SIRT2) exerts a protective effects role in TBI, although the underlying mechanism of this protection remains unclear. In the present study, we tested the hypothesis that that SIRT2 mitigates TBI by regulating p53-mediated ferroptosis. To model TBI in mice, we used the controlled cortical impact (CCI) injury method. We found that ferroptosis was significantly activated by CCI, and peaked 3 days following CCI, as evidenced by upregulation of GPX4 and SLC7A11, increased content of decreases glutathione, lipid peroxidation, malondialdehyde and ferrous ion. Inhibition of ferroptosis significantly alleviated neurological indications and brain edema. In addition, knockout of p53 significantly blocked ferroptosis following CCI. Furthermore, we found that inhibition of SIRT2 upregulated the acetylation of p53, as well as p53 expression, and exacerbated ferroptosis following CCI. Interestingly, knockout of p53 rescued the SIRT2 inhibition-induced exacerbation of ferroptosis. These findings indicate that p53-mediated ferroptosis contributes to the pathogenesis of TBI. Furthermore, we demonstrate that SIRT2 exerts a neuroprotective effect against TBI by suppressing p53-mediated ferroptosis.