Amelioration of Cognitive and Behavioral Deficits after Traumatic Brain Injury in Coagulation Factor XII Deficient Mice.

Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII-/- mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII-/- mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII-/- mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII-/- mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII-/- mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.

Posttraumatic learning deficits correlate with initial trauma severity and chronic cellular reactions after closed head injury in male mice.

Traumatic brain injury (TBI) is often associated with sustained attention and memory deficits. As persisting neuroinflammation and neurodegeneration may contribute to posttraumatic psychomotor dysfunction, we studied the relationship of brain cellular reactions three months after a weight-drop closed head injury in male mice with posttraumatic learning and memory using automated home-cage monitoring of socially housed mice in IntelliCages as well as tests for locomotor activity, anxiety and forepaw fine motor skills. One month after TBI, deficits in place learning and cognitive flexibility in reverse learning were clearly detectable in IntelliCages and these memory deficits correlated with the initial trauma severity on the functional neuroscore. While sucrose preference or its extinction were not influenced by TBI, traumatized mice performed significantly worse in a complex episodic memory learning task. In consecutive locomotor and forepaw skilled use tests, posttraumatic hyperactivity and impairment of contralateral paw use were evident. Analysis of cellular reactions to TBI three months after injury in selected defined regions of interest in the immediate lesion, ipsi- and contralateral frontoparietal cortex and hippocampus revealed a persistent microgliosis and astrogliosis which were accompanied by iron-containing macrophages and myelin degradation in the lesion area as well as with axonal damage in the neighboring cortical regions. Microglial and astroglial reactions in cortex showed a positive correlation with the initial trauma severity and a negative correlation with the spatial and episodic memory indicating a role of brain inflammatory reactions in posttraumatic memory deficits.

Skull Fractures Induce Neuroinflammation and Worsen Outcomes after Closed Head Injury in Mice.

The weight-drop model is used widely to replicate closed-head injuries in mice; however, the histopathological and functional outcomes may vary significantly between laboratories. Because skull fractures are reported to occur in this model, we aimed to evaluate whether these breaks may influence the variability of the weight-drop (WD) model. Male Swiss Webster mice underwent WD injury with either a 2 or 5 mm cone tip, and behavior was assessed at 2 h and 24 h thereafter using the neurological severity score. The expression of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 genes was measured at 12 h and 1, 3, and 14 days after injury. Before the injury, micro-computed tomography (micro-CT) was performed to quantify skull thickness at the impact site. With a conventional tip diameter of 2 mm, 33% of mice showed fractures of the parietal bone; the 5 mm tip produced only 10% fractures. Compared with mice without fractures, mice with fractures had a severity-dependent worse functional outcome and a more pronounced upregulation of inflammatory genes in the brain. Older mice were associated with thicker parietal bones and were less prone to skull fractures. In addition, mice that underwent traumatic brain injury (TBI) with skull fracture had macroscopic brain damage because of skull depression. Skull fractures explain a considerable proportion of the variability observed in the WD model in mice-i.e., mice with skull fractures have a much stronger inflammatory response than do mice without fractures. Using older mice with thicker skull bones and an impact cone with a larger diameter reduces the rate of skull fractures and the variability in this very useful closed-head TBI model.

How is the formation of microthrombi after traumatic brain injury linked to inflammation?

Traumatic brain injury (TBI) is characterized by mechanical disruption of brain tissue due to an external force and by subsequent secondary injury. Secondary brain injury events include inflammatory responses and the activation of coagulation resulting in microthrombi formation in the brain vasculature. Recent research suggests that these mechanisms do not work independently. There is strong evidence that FXII and platelet activation connects both, inflammation and the formation of microthrombi. This review summarizes the current knowledge on posttraumatic microthrombus formation and its link to inflammation.

Alleviation of secondary brain injury, posttraumatic inflammation, and brain edema formation by inhibition of factor XIIa.

BACKGROUND: Traumatic brain injury (TBI) is a devastating neurological condition and a frequent cause of permanent disability. Posttraumatic inflammation and brain edema formation, two pathological key events contributing to secondary brain injury, are mediated by the contact-kinin system. Activation of this pathway in the plasma is triggered by activated factor XII. Hence, we set out to study in detail the influence of activated factor XII on the abovementioned pathophysiological features of TBI. METHODS: Using a cortical cryogenic lesion model in mice, we investigated the impact of genetic deficiency of factor XII and inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused Infestin-4 on the release of bradykinin, the brain lesion size, and contact-kinin system-dependent pathological events. We determined protein levels of bradykinin, intracellular adhesion molecule-1, CC-chemokine ligand 2, and interleukin-1ß by enzyme-linked immunosorbent assays and mRNA levels of genes related to inflammation by quantitative real-time PCR. Brain lesion size was determined by tetrazolium chloride staining. Furthermore, protein levels of the tight junction protein occludin, integrity of the blood-brain barrier, and brain water content were assessed by Western blot analysis, extravasated Evans Blue dye, and the wet weight-dry weight method, respectively. Infiltration of neutrophils and microglia/activated macrophages into the injured brain lesions was quantified by immunohistological stainings. RESULTS: We show that both genetic deficiency of factor XII and inhibition of activated factor XII in mice diminish brain injury-induced bradykinin release by the contact-kinin system and minimize brain lesion size, blood-brain barrier leakage, brain edema formation, and inflammation in our brain injury model. CONCLUSIONS: Stimulation of bradykinin release by activated factor XII probably plays a prominent role in expanding secondary brain damage by promoting brain edema formation and inflammation. Pharmacological blocking of activated factor XII could be a useful therapeutic principle in the treatment of TBI-associated pathologic processes by alleviating posttraumatic inflammation and brain edema formation.

Combined [(18)F]DPA-714 micro-positron emission tomography and autoradiography imaging of microglia activation after closed head injury in mice.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability. Neuroinflammation contributes to acute damage after TBI and modulates long-term evolution of degenerative and regenerative responses to injury. The aim of the present study was to evaluate the relationship of microglia activation to trauma severity, brain energy metabolism, and cellular reactions to injury in a mouse closed head injury model using combined in vivo PET imaging, ex vivo autoradiography, and immunohistochemistry. METHODS: A weight-drop closed head injury model was used to produce a mixed diffuse and focal TBI or a purely diffuse mild TBI (mTBI) in C57BL6 mice. Lesion severity was determined by evaluating histological damage and functional outcome using a standardized neuroscore (NSS), gliosis, and axonal injury by immunohistochemistry. Repeated intra-individual in vivo µPET imaging with the specific 18-kDa translocator protein (TSPO) radioligand [(18)F]DPA-714 was performed on day 1, 7, and 16 and [(18)F]FDG-µPET imaging for energy metabolism on days 2-5 after trauma using freshly synthesized radiotracers. Immediately after [(18)F]DPA-714-µPET imaging on days 7 and 16, cellular identity of the [(18)F]DPA-714 uptake was confirmed by exposing freshly cut cryosections to film autoradiography and successive immunostaining with antibodies against the microglia/macrophage marker IBA-1. RESULTS: Functional outcome correlated with focal brain lesions, gliosis, and axonal injury. [(18)F]DPA-714-µPET showed increased radiotracer uptake in focal brain lesions on days 7 and 16 after TBI and correlated with reduced cerebral [(18)F]FDG uptake on days 2-5, with functional outcome and number of IBA-1 positive cells on day 7. In autoradiography, [(18)F]DPA-714 uptake co-localized with areas of IBA1-positive staining and correlated strongly with both NSS and the number of IBA1-positive cells, gliosis, and axonal injury. After mTBI, numbers of IBA-1 positive cells with microglial morphology increased in both brain hemispheres; however, uptake of [(18)F]DPA-714 was not increased in autoradiography or in µPET imaging. CONCLUSIONS: [(18)F]DPA-714 uptake in µPET/autoradiography correlates with trauma severity, brain metabolic deficits, and microglia activation after closed head TBI.

Targeting coagulation factor XII as a novel therapeutic option in brain trauma.

OBJECTIVE: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury. METHODS: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury. RESULTS: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage. INTERPRETATION: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. Ann Neurol 2016;79:970-982.

Role of the kallikrein-kinin system in traumatic brain injury.

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Despite improvements in acute intensive care, there are currently no specific therapies to ameliorate the effects of TBI. Successful therapeutic strategies for TBI should target multiple pathophysiologic mechanisms that occur at different stages of brain injury. The kallikrein-kinin system is a promising therapeutic target for TBI as it mediates key pathologic events of traumatic brain damage, such as edema formation, inflammation, and thrombosis. Selective and specific kinin receptor antagonists and inhibitors of plasma kallikrein and coagulation factor XII have been developed, and have already shown therapeutic efficacy in animal models of stroke and TBI. However, conflicting preclinical evaluation, as well as limited and inconclusive data from clinical trials in TBI, suggests that caution should be taken before transferring observations made in animals to humans. This review summarizes current evidence on the pathologic significance of the kallikrein-kinin system during TBI in animal models and, where available, the experimental findings are compared with human data.

C1-Inhibitor protects from focal brain trauma in a cortical cryolesion mice model by reducing thrombo-inflammation.

Traumatic brain injury (TBI) induces a strong inflammatory response which includes blood-brain barrier damage, edema formation and infiltration of different immune cell subsets. More recently, microvascular thrombosis has been identified as another pathophysiological feature of TBI. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is activated in different neurological diseases. C1-Inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-Inhibitor in a model of TBI. Male and female C57BL/6 mice were subjected to cortical cryolesion and treated with C1-Inhibitor after 1 h. Lesion volumes were assessed between day 1 and day 5 and blood-brain barrier damage, thrombus formation as well as the local inflammatory response were determined post TBI. Treatment of male mice with 15.0 IU C1-Inhibitor, but not 7.5 IU, 1 h after cryolesion reduced lesion volumes by ~75% on day 1. This protective effect was preserved in female mice and at later stages of trauma. Mechanistically, C1-Inhibitor stabilized the blood-brain barrier and decreased the invasion of immune cells into the brain parenchyma. Moreover, C1-Inhibitor had strong antithrombotic effects. C1-Inhibitor represents a multifaceted anti-inflammatory and antithrombotic compound that prevents traumatic neurodegeneration in clinically meaningful settings.

FTY720 does not protect from traumatic brain injury in mice despite reducing posttraumatic inflammation.

Inflammation is a pathological hallmark of traumatic brain injury (TBI). Recent evidence suggests that immune cells such as lymphocytes are of particular relevance for lesion development after TBI. FTY720, a sphingosine-1-phosphate (S1P) receptor modulator, sequesters T lymphocytes in lymphoid organs and has been shown to improve outcome in a variety of neurological disease models. We investigated the mode of FTY720 action in models of TBI. Focal cortical cryolesion was induced in C57BL/6 mice treated with FTY720 (1mg/kg) or vehicle immediately before injury. Lesion size was assessed 24h later. Immune cells in the blood and brain were counted by flow cytometry and immunocytochemistry. The integrity of the blood-brain barrier was analyzed using Evans Blue dye. To validate the findings in a diffuse brain trauma model, FTY720-treated mice and controls were subjected to weight drop contusion injury and neurological deficits were assessed until day 7. As expected FTY720 significantly lowered the numbers of circulating lymphocytes and attenuated the invasion of immune cells into the damaged brain parenchyma. However, FTY720 was unable to improve lesion size or functional outcome in both trauma models at either stage, i.e. acute vs chronic. Accordingly, the extent of blood-brain barrier disruption and neuronal apoptosis was similar between FTY720-treated mice and controls. We conclude that pharmacological S1P receptor modulation is an unfavorable strategy to combat TBI. Moreover, our findings put into perspective the pathophysiological relevance of inflammatory cells in traumatic neurodegeneration.

Ischemic stroke and traumatic brain injury: the role of the kallikrein-kinin system.

Acute ischemic stroke and traumatic brain injury are a major cause of mortality and morbidity. Due to the paucity of therapies, there is a pressing clinical demand for new treatment options. Successful therapeutic strategies for these conditions must target multiple pathophysiological mechanisms occurring at different stages of brain injury. In this respect, the kallikrein-kinin system is an ideal target linking key pathological hallmarks of ischemic and traumatic brain damage such as edema formation, inflammation, and thrombosis. In particular, the kinin receptors, plasma kallikrein, and coagulation factor XIIa are highly attractive candidates for pharmacological development, as kinin receptor antagonists or inhibitors of plasma kallikrein and coagulation factor XIIa are neuroprotective in animal models of stroke and traumatic brain injury. Nevertheless, conflicting preclinical evaluation as well as limited and inconclusive data from clinical trials suggest caution when transferring observations made in animals into the human situation. This review summarizes current evidence on the pathological significance of the kallikrein-kinin system during ischemic and traumatic brain damage, with a particular focus on experimental data derived from animal models. Experimental findings are also compared with human data if available, and potential therapeutic implications are discussed.

A modified double injection model of cisterna magna for the study of delayed cerebral vasospasm following subarachnoid hemorrhage in rats.

Delayed cerebral vasospasm following subarachnoid hemorrhage (SAH) is a serious medical complication, characterized by constriction of cerebral arteries leading to varying degrees of cerebral ischemia. Numerous clinical and experimental studies have been performed in the last decades; however, the pathophysiologic mechanism of cerebral vasospasm after SAH still remains unclear. Among a variety of experimental SAH models, the double hemorrhage rat model involving direct injection of autologous arterial blood into the cisterna magna has been used most frequently for the study of delayed cerebral vasospasm following SAH in last years. Despite the simplicity of the technique, the second blood injection into the cisterna magna may result in brainstem injury leading to high mortality. Therefore, a modified double hemorrhage model of cisterna magna has been developed in rat recently. We describe here step by step the surgical technique to induce double SAH and compare the degree of vasospasm with other cisterna magna rat models using histological assessment of the diameter and cross-sectional area of the basilar artery.

C1-inhibitor protects from brain ischemia-reperfusion injury by combined antiinflammatory and antithrombotic mechanisms.

BACKGROUND AND PURPOSE: Inflammation and thrombosis are pathophysiological hallmarks of ischemic stroke still unamenable to therapeutic interventions. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is involved in stroke development. C1-inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-inhibitor in models of ischemic stroke. METHODS: Male and female C57Bl/6 mice and rats of different ages were subjected to middle cerebral artery occlusion and treated with C1-inhibitor after 1 hour or 6 hours. Infarct volumes and functional outcomes were assessed between day 1 and day 7, and findings were validated by magnetic resonance imaging. Blood-brain barrier damage, thrombus formation, and the local inflammatory response were determined poststroke. RESULTS: Treatment with 15.0 U C1-inhibitor, but not 7.5 U, 1 hour after stroke reduced infarct volumes by ≈60% and improved clinical scores in mice of either sex on day 1. This protective effect was preserved at later stages of infarction as well as in elderly mice and in another species, ie, rats. Delayed C1-inhibitor treatment still improved clinical outcome. Blood-brain barrier damage, edema formation, and inflammation were significantly lower compared with controls. Moreover, C1-inhibitor showed strong antithrombotic effects. CONCLUSIONS: C1-inhibitor is a multifaceted antiinflammatory and antithrombotic compound that protects from ischemic neurodegeneration in clinically meaningful settings.

Blocking of bradykinin receptor B1 protects from focal closed head injury in mice by reducing axonal damage and astroglia activation.

The two bradykinin receptors B1R and B2R are central components of the kallikrein-kinin system with different expression kinetics and binding characteristics. Activation of these receptors by kinins triggers inflammatory responses in the target organ and in most situations enhances tissue damage. We could recently show that blocking of B1R, but not B2R, protects from cortical cryolesion by reducing inflammation and edema formation. In the present study, we investigated the role of B1R and B2R in a closed head model of focal traumatic brain injury (TBI; weight drop). Increased expression of B1R in the injured hemispheres of wild-type mice was restricted to the later stages after brain trauma, i.e. day 7 (P<0.05), whereas no significant induction could be observed for the B2R (P>0.05). Mice lacking the B1R, but not the B2R, showed less functional deficits on day 3 (P<0.001) and day 7 (P<0.001) compared with controls. Pharmacological blocking of B1R in wild-type mice had similar effects. Reduced axonal injury and astroglia activation could be identified as underlying mechanisms, while inhibition of B1R had only little influence on the local inflammatory response in this model. Inhibition of B1R may become a novel strategy to counteract trauma-induced neurodegeneration.

Focal brain trauma in the cryogenic lesion model in mice.

The method to induce unilateral cryogenic lesions was first described in 1958 by Klatzo. We describe here an adaptation of this model that allows reliable measurement of lesion volume and vasogenic edema by 2, 3, 5-triphenyltetrazolium chloride-staining and Evans blue extravasation in mice. A copper or aluminium cylinder with a tip diameter of 2.5 mm is cooled with liquid nitrogen and placed on the exposed skull bone over the parietal cortex (coordinates from bregma: 1.5 mm posterior, 1.5 mm lateral). The tip diameter and the contact time between the tip and the parietal skull determine the extent of cryolesion. Due to an early damage of the blood brain barrier, the cryogenic cortical injury is characterized by vasogenic edema, marked brain swelling, and inflammation. The lesion grows during the first 24 hours, a process involving complex interactions between endothelial cells, immune cells, cerebral blood flow, and the intracranial pressure. These contribute substantially to the damage from the initial injury. The major advantage of the cryogenic lesion model is the circumscribed and highly reproducible lesion size and location.

An experimental protocol for mimicking pathomechanisms of traumatic brain injury in mice.

Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.

Experimental traumatic brain injury.

Traumatic brain injury, a leading cause of death and disability, is a result of an outside force causing mechanical disruption of brain tissue and delayed pathogenic events which collectively exacerbate the injury. These pathogenic injury processes are poorly understood and accordingly no effective neuroprotective treatment is available so far. Experimental models are essential for further clarification of the highly complex pathology of traumatic brain injury towards the development of novel treatments. Among the rodent models of traumatic brain injury the most commonly used are the weight-drop, the fluid percussion, and the cortical contusion injury models. As the entire spectrum of events that might occur in traumatic brain injury cannot be covered by one single rodent model, the design and choice of a specific model represents a major challenge for neuroscientists. This review summarizes and evaluates the strengths and weaknesses of the currently available rodent models for traumatic brain injury.

Control of flagellar gene regulation in Legionella pneumophila and its relation to growth phase.

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.

Two small ncRNAs jointly govern virulence and transmission in Legionella pneumophila.

To transit from intra- to extracellular environments, Legionella pneumophila differentiates from a replicative/non-virulent to a transmissive/virulent form using the two-component system LetA/LetS and the global repressor protein CsrA. While investigating how both regulators act co-ordinately we characterized two ncRNAs, RsmY and RsmZ, that link the LetA/LetS and CsrA regulatory networks. We demonstrate that LetA directly regulates their expression and show that RsmY and RsmZ are functional in Escherichia coli and are able to bind CsrA in vitro. Single mutants have no (ΔrsmY) or a little (ΔrsmZ) impact on virulence, but the ΔrsmYZ strain shows a drastic defect in intracellular growth in Acanthamoeba castellanii and THP-1 monocyte-derived macrophages. Analysis of the transcriptional programmes of the ΔletA, ΔletS and ΔrsmYZ strains revealed that the switch to the transmissive phase is partially blocked. One major difference between the ΔletA, ΔletS and ΔrsmYZ strains was that the latter synthesizes flagella. Taken together, LetA activates transcription of RsmY and RsmZ, which sequester CsrA and abolish its post-transcriptional repressive activity. However, the RsmYZ-CsrA pathway appears not to be the main or only regulatory circuit governing flagella synthesis. We suggest that rather RpoS and LetA, by influencing LetE and probably cyclic-di-GMP levels, regulate motility in L. pneumophila.