Fredrik Clausen

Neutralization of interleukin-1β modifies the inflammatory response and improves histological and cognitive outcome following traumatic brain injury in mice

Interleukin‐1β (IL‐1β) may play a central role in the inflammatory response following traumatic brain injury (TBI). We subjected 91 mice to controlled cortical impact (CCI) brain injury or sham injury. Beginning 5 min post‐injury, the IL‐1β neutralizing antibody IgG2a/k (1.5 μg/mL) or control antibody was infused at a rate of 0.25 μL/h into the contralateral ventricle for up to 14 days using osmotic minipumps. Neutrophil and T‐cell infiltration and microglial activation was evaluated at days 1–7 post‐injury. Cognition was assessed using Morris water maze, and motor function using rotarod and cylinder tests. Lesion volume and hemispheric tissue loss were evaluated at 18 days post‐injury. Using this treatment strategy, cortical and hippocampal tissue levels of IgG2a/k reached 50 ng/mL, sufficient to effectively inhibit IL‐1βin vitro. IL‐1β neutralization attenuated the CCI‐induced cortical and hippocampal microglial activation (P < 0.05 at post‐injury days 3 and 7), and cortical infiltration of neutrophils (P < 0.05 at post‐injury day 7). There was only a minimal cortical infiltration of activated T‐cells, attenuated by IL‐1β neutralization (P < 0.05 at post‐injury day 7). CCI induced a significant deficit in neurological motor and cognitive function, and caused a loss of hemispheric tissue (P < 0.05). In brain‐injured animals, IL‐1β neutralizing treatment resulted in reduced lesion volume, hemispheric tissue loss and attenuated cognitive deficits (P < 0.05) without influencing neurological motor function. Our results indicate that IL‐1β is a central component in the post‐injury inflammatory response that, in view of the observed positive neuroprotective and cognitive effects, may be a suitable pharmacological target for the treatment of TBI.

Free Radical Scavenger Posttreatment Improves Functional and Morphological Outcome after Fluid Percussion Injury in the Rat

Reactive oxygen species (ROS) are thought to contribute to the secondary injury process after traumatic brain injury (TBI). ROS scavenging compounds have shown neuroprotective properties in various models of experimental brain injury, including TBI. Administration of nitrone radical scavengers has emerged as a promising pharmacological concept in focal experimental ischemia due to their low toxicity and neuroprotective properties, with a time window of several hours. The aim of this study was to test the neuroprotective efficacy of two nitrones, the readily blood-brain barrier (BBB) penetrating alpha-phenyl-N-tert-butyl nitrone (PBN) and the poorly BBB penetrating sulfo-derivative, 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) after moderate (2.20-2.45 atm) lateral fluid percussion injury (FPI) in rats. Twenty-six rats received a 24-h intravenous infusion (30 mg/kg/h) of saline, PBN, or an equimolar dose of S-PBN beginning 30 min after FPI. Eight sham-operated animals were used as controls. Cognitive function was assessed using the Morris Water Maze at day 11-15 after TBI, neurological status at day 1, 4, and 8 and morphological outcome at day 15. PBN and S-PBN treatment significantly reduced the loss of ipsilateral hemispheric tissue whereas only S-PBN tended to reduce the cortical lesion volume. PBN treatment caused a significant improvement in the neurological score as compared to saline-treated animals, while S-PBN alone attenuated the cognitive deficit. Our results suggest that nitrone radical scavengers are neuroprotective when administered 30 min after FPI in rats. Differences in pharmacokinetics may account for the observed individual neuroprotective profiles of the two nitrones.

Neutralization of Interleukin-1β Reduces Cerebral Edema and Tissue loss and Improves Late Cognitive Outcome Following Traumatic Brain Injury in Mice

Increasing evidence suggests that interleukin‐1β (IL‐1β) is a key mediator of the inflammatory response following traumatic brain injury (TBI). Recently, we showed that intracerebroventricular administration of an IL‐1β‐neutralizing antibody was neuroprotective following TBI in mice. In the present study, an anti‐IL‐1β antibody or control antibody was administered intraperitoneally following controlled cortical injury (CCI) TBI or sham injury in 105 mice and we extended our histological, immunological and behavioral analysis. First, we demonstrated that the treatment antibody reached target brain regions of brain‐injured animals in high concentrations (> 11 nm) remaining up to 8 days post‐TBI. At 48 h post‐injury, the anti‐IL‐1β treatment attenuated the TBI‐induced hemispheric edema (P < 0.05) but not the memory deficits evaluated using the Morris water maze (MWM). Neutralization of IL‐1β did not influence the TBI‐induced increases (P < 0.05) in the gene expression of the Ccl3 and Ccr2 chemokines, IL‐6 or Gfap. Up to 20 days post‐injury, neutralization of IL‐1β was associated with improved visuospatial learning in the MWM, reduced loss of hemispheric tissue and attenuation of the microglial activation caused by TBI (P < 0.05). Motor function using the rotarod and cylinder tests was not affected by the anti‐IL‐1β treatment. Our results suggest an important negative role for IL‐1β in TBI. The improved histological and behavioral outcome following anti‐IL‐1β treatment also implies that further exploration of IL‐1β‐neutralizing compounds as a treatment option for TBI patients is warranted.

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

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 Evans 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 Evans 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.

Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice

Oligomeric assemblies of amyloid‐β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long‐term potentiation. The tg‐ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg‐ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg‐ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg‐ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg‐ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg‐ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age‐dependent manner in tg‐ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.

Monitoring of Reactive Oxygen Species Production after Traumatic Brain Injury in Rats with Microdialysis and the 4-Hydroxybenzoic Acid Trapping Method

The detection of reactive oxygen species (ROS) after traumatic brain injury (TBI) is based on indirect methods due to the high reactivity and short half-life of ROS in biological tissue. The commonly used salicylate trapping method has several disadvantages making it unsuitable for human use. We have evaluated 4-hydroxybenzoic acid (4-HBA) together with microdialysis (MD) in the rat as an alternative method. 4-HBA forms one stable adduct, 3,4-dihydroxybenzoic acid (3,4-DHBA), when reacting with ROS and has not previously been used together with MD after TBI. Twenty-seven rats were used for the assessment of 3,4-DHBA production as an indicator of ROS formation in a controlled contusion injury model using intracerebral MD with 3 mM 4-HBA in the perfusate. For comparison, salicylate trapping was used in eight rats. TBI caused a 250% increase of 3,4-DHBA that peaked at 30 min after injury in severely injured rats and remained significantly elevated as compared to baseline for 90 min after trauma. The mild injury level caused a 100% increase in 3,4-DHBA formation at 30 min after the injury. When the MD probe was placed in the perimeter of the injury site, no significant increase in ROS formation occurred. Salicylate trapping showed a similar increase in adduct formation after severe injury. In addition, high cortical concentrations of 4-HBA and salicylate were found. It is concluded that microdialysis with 4-HBA as a trapping agent appears to be a useful method for ROS detection in the rat with a potential clinical utility.

Effects of the Nitrone Radical Scavengers PBN and S-PBN on In vivo Trapping of Reactive Oxygen Species after Traumatic Brain Injury in Rats:

In previous studies, the authors showed that the nitrone radical scavenger α-phenyl-N-tert-butyl nitrone (PBN) and its sulfo-derivative, 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN), attenuated cognitive disturbance and reduced tissue damage after traumatic brain injury (TBI) in rats. In the current study, the production of reactive oxygen species (ROS) after TBI was monitored with microdialysis and the 4-hydroxybenzoic acid (4-HBA) trapping method. A single dose of PBN (30 mg/kg) or an equimolar dose of S-PBN (47 mg/kg) was administered intravenously 30 minutes before a controlled cortical contusion injury in rats. Plasma and brain tissue drug concentrations were analyzed at the end of the microdialysis experiment (3 hours after injury) and, in a separate experiment with S-PBN, at 30 and 60 minutes after injury. Traumatic brain injury caused a significant increase in ROS formation that lasted for 60 minutes after the injury as evidenced by increased 3,4-dihydroxybenzoic acid (3,4-DHBA) concentrations in the dialysate. PBN and S-PBN equally and significantly attenuated the posttraumatic increase in 3,4-DHBA formation. High PBN concentrations were found bilaterally in brain tissue up to 3 hours after injury. In contrast, S-PBN was rapidly cleared from the circulation and was not detectable in brain at 30 minutes after injury or at any later time point. The results suggest that scavenging of ROS after TBI may contribute to the neuroprotective properties observed with nitrone spin-trapping agents. S-PBN, which remained undetectable even in traumatized brain tissue, reduced ROS production to the same extent as PBN that readily crossed the blood–brain barrier. This finding supports an important role for ROS production at the blood–endothelial interface in TBI.

alpha-Phenyl-tert-N-butyl nitrone (PBN) improves functional and morphological outcome after cortical contusion injury in the rat.

Summary α-Phenyl-tert-N-butyl nitrone (PBN), a potent reactive oxygen species (ROS) scavenger, has shown robust neuroprotective properties in several models of acute brain injury, although not previously evaluated in traumatic brain injury (TBI). In this study, we assessed the potential efficacy of PBN in a weight drop model producing a controlled cortical contusion. Sham operation, mild or severe injury was induced in intubated and ventilated rats and functional and morphological outcome was used as end-points at two weeks post-injury. In the trauma groups, saline or PBN (30 mg/kg) was injected as an intravenous bolus 30 minutes prior to injury. At day 11–15 post-injury, cognitive disturbance was assessed using the Morris Water Maze (MWM) and estimation of lesion volume and hemispheric loss of tissue was made. No change in MWM performance were found in either of the mildly traumatized groups as compared to uninjured controls. In contrast, a significant decrease in total mean latency and increase in path length in the severely traumatized rats were found. PBN-treatment significantly improved MWM performance as compared to saline treatment at the severe injury level (p<0.05). The mild injury level caused a discrete atrophy of the ipsilateral cortex with no effect of PBN treatment. The severe injury caused a substantial loss of ipsilateral hemispheric tissue and a large cortical cavitation. PBN pre-treatment significantly reduced the lesion volume and reduced hemispheric loss of tissue at this injury level (p<0.05). Our results support the involvement of ROS in the injury process contributing to the tissue loss and cognitive disturbance after TBI. The potential clinical utility of PBN will have to be assessed using a post-injury dosing regime.

Paradoxical Increase in Neuronal DNA Fragmentation after Neuroprotective Free Radical Scavenger Treatment in Experimental Traumatic Brain Injury

The mechanisms and role of nerve cell death after traumatic brain injury (TBI) are not fully understood. The authors investigated the effect of pretreatment with the oxygen free radical spin trap α-phenyl-N-tert-butyl-nitrone (PBN) on the number of neurons undergoing apoptosis after TBI in rats. Apoptotic cells were identified by the TUNEL method combined with the nuclear stain, Hoechst 33258, and immunohistochemistry for the active form of caspase-3. Numerous neurons became positive for activated caspase 3 and TUNEL in the cortex at 24 hours after injury, suggesting ongoing biochemical apoptosis. In PBN-treated rats, a significantly greater number of cells were found to be TUNEL positive at 24 hours compared with controls. However, PBN treatment resulted in a reduced cortical lesion volume and improved behavioral outcome two weeks after injury. The authors conclude that a treatment producing an increase in DNA fragmentation in the early phase may be compatible with an overall beneficial effect on outcome after TBI. This should be considered in the screening process for future neuroprotective remedies.

Interstitial F2-Isoprostane 8-Iso-PGF2α As a Biomarker of Oxidative Stress after Severe Human Traumatic Brain Injury

Oxidative stress is a major contributor to the secondary injury process after experimental traumatic brain injury (TBI). The importance of oxidative stress in the pathobiology of human TBI is largely unknown. The F(2)-isoprostane 8-iso-prostaglandin F(2α) (8-iso-PGF(2α)), synthesized in vivo through non-enzymatic free radical catalyzed peroxidation of arachidonic acid, is a widely used biomarker of oxidative stress in multiple disease states, including TBI and cerebral ischemia/reperfusion. Our hypothesis is that harvesting of biomarkers directly in the injured brain by cerebral microdialysis (MD) is advantageous because of its high spatial and temporal resolution compared to blood or cerebrospinal fluid sampling. The aim of this study was to test the feasibility of measuring 8-iso-PGF(2α) in MD, ventricular cerebrospinal fluid (vCSF), and plasma samples collected from patients with severe TBI, and to compare the MD signals with MD-glycerol, implicated as a biomarker of oxidative stress, as well as MD-glutamate, a biomarker of excitotoxicity. Six patients (4 men, 2 women) were included in the study, three of whom had a focal/mixed TBI, and three a diffuse axonal injury (DAI). Following the bedside analysis of routine MD biomarkers (glucose, lactate:pyruvate ratio, glycerol, and glutamate), two 12-h MD samples per day were used to analyze 8-iso-PGF(2α) from 24 h up to 8 days post-injury. The interstitial levels of 8-iso-PGF(2α) were markedly higher than the levels obtained from plasma and vCSF (p<0.05), supporting our hypothesis. The MD-8-iso-PGF(2α) levels correlated strongly (p<0.05) with MD-glycerol and MD-glutamate, which are widely used biomarkers of membrane phospholipid degradation/oxidative stress and excitotoxicity, respectively. This study demonstrates the feasibility of analyzing 8-iso-PGF(2α) in MD samples from the human brain. Our results support a close relationship between oxidative stress and excitotoxicity following human TBI. MD-8-iso-PGF(2α) in combination with MD-glycerol may be useful biomarkers of oxidative stress in the neurointensive care setting.