Clara Luh

Influence of Age on Brain Edema Formation, Secondary Brain Damage and Inflammatory Response after Brain Trauma in Mice

After traumatic brain injury (TBI) elderly patients suffer from higher mortality rate and worse functional outcome compared to young patients. However, experimental TBI research is primarily performed in young animals. Aim of the present study was to clarify whether age affects functional outcome, neuroinflammation and secondary brain damage after brain trauma in mice. Young (2 months) and old (21 months) male C57Bl6N mice were anesthetized and subjected to a controlled cortical impact injury (CCI) on the right parietal cortex. Animals of both ages were randomly assigned to 15 min, 24 h, and 72 h survival. At the end of the observation periods, contusion volume, brain water content, neurologic function, cerebral and systemic inflammation (CD3+ T cell migration, inflammatory cytokine expression in brain and lung, blood differential cell count) were determined. Old animals showed worse neurological function 72 h after CCI and a high mortality rate (19.2%) compared to young (0%). This did not correlate with histopathological damage, as contusion volumes were equal in both age groups. Although a more pronounced brain edema formation was detected in old mice 24 hours after TBI, lack of correlation between brain water content and neurological deficit indicated that brain edema formation is not solely responsible for age-dependent differences in neurological outcome. Brains of old naïve mice were about 8% smaller compared to young naïve brains, suggesting age-related brain atrophy with possible decline in plasticity. Onset of cerebral inflammation started earlier and primarily ipsilateral to damage in old mice, whereas in young mice inflammation was delayed and present in both hemispheres with a characteristic T cell migration pattern. Pulmonary interleukin 1β expression was up-regulated after cerebral injury only in young, not aged mice. The results therefore indicate that old animals are prone to functional deficits and strong ipsilateral cerebral inflammation without major differences in morphological brain damage compared to young.

Volatile Anesthetics Influence Blood-Brain Barrier Integrity by Modulation of Tight Junction Protein Expression in Traumatic Brain Injury

Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonula occludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER) in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI). In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU) volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate adverse or beneficial effects of volatile anesthetics on patients at risk for cerebral edema.

Pioglitazone Reduces Secondary Brain Damage after Experimental Brain Trauma by PPAR-γ-Independent Mechanisms

Inflammatory and ischemic processes contribute to the development of secondary brain damage after mechanical brain injury. Recent data suggest that thiazolidinediones (TZDs), a class of drugs approved for the treatment of non-insulin-dependent diabetes mellitus, effectively reduces inflammation and brain lesion by stimulation of the peroxisome proliferator-activated receptor-γ (PPAR-γ). The present study investigates the influence of the TZD pioglitazone and rosiglitazone on inflammation and secondary brain damage after experimental traumatic brain injury (TBI). A controlled cortical impact (CCI) injury was induced in male C57BL/6 mice to investigate following endpoints: (1) mRNA expression of PPAR-γ and PPAR-γ target genes (LPL, GLT1, and IRAP/Lnpep), and inflammatory markers (TNF-α, IL-1β, IL-6, and iNOS), at 15 min, 3 h, 6 h, 12 h, and 24 h post-trauma; (2) contusion volume, neurological function, and gene expression after 24 h in mice treated with pioglitazone (0.5 and 1 mg/kg) or rosiglitazone (5 and 10 mg/kg IP at 30 min post-trauma); and (3) the role of PPAR-γ to mediate protection was determined in animals treated with pioglitazone, the PPAR-γ inhibitor T0070907, and a combination of both. Inflammatory marker genes, but not PPAR-γ gene expression, was upregulated after trauma. Pioglitazone reduced the histological damage and inflammation in a dose-dependent fashion. In contrast, rosiglitazone failed to suppress inflammation and histological damage. PPAR-γ and PPAR-γ target gene expression was not induced by pioglitazone and rosiglitazone. In line with these results, pioglitazone-mediated protection was not reversed by T0070907. The results indicate that the neuroprotective effects of pioglitazone are not solely related to PPAR-γ-dependent mechanisms.

Inhibition of myosin light chain kinase reduces brain edema formation after traumatic brain injury

J. Neurochem. (2009) 112, 1015–1025.

Single Administration of Tripeptide α-MSH(11–13) Attenuates Brain Damage by Reduced Inflammation and Apoptosis after Experimental Traumatic Brain Injury in Mice

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11–13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11–13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11–13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11–13) injected mice. The expression of the inflammatory markers TNF-α and IL-1β as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11–13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.

Influence of a Brief Episode of Anesthesia during the Induction of Experimental Brain Trauma on Secondary Brain Damage and Inflammation

It is unclear whether a single, brief, 15-minute episode of background anesthesia already modulates delayed secondary processes after experimental brain injury. Therefore, this study was designed to characterize three anesthesia protocols for their effect on molecular and histological study endpoints. Mice were randomly separated into groups that received sevoflurane (sevo), isoflurane (iso) or an intraperitoneal anesthetic combination (midazolam, fentanyl and medetomidine; comb) prior to traumatic brain injury (controlled cortical impact, CCI; 8 m/s, 1 mm impact depth, 3 mm diameter). Twenty-four hours after insult, histological brain damage, neurological function (via neurological severity score), cerebral inflammation (via real-time RT-PCR for IL6, COX-2, iNOS) and microglia (via immunohistochemical staining for Iba1) were determined. Fifteen minutes after CCI, the brain contusion volume did not differ between the anesthetic regimens (sevo = 17.9±5.5 mm3; iso = 20.5±3.7 mm3; comb = 19.5±4.6 mm3). Within 24 hours after injury, lesion size increased in all groups (sevo = 45.3±9.0 mm3; iso = 31.5±4.0 mm3; comb = 44.2±6.2 mm3). Sevo and comb anesthesia resulted in a significantly larger contusion compared to iso, which was in line with the significantly better neurological function with iso (sevo = 4.6±1.3 pts.; iso = 3.9±0.8 pts.; comb = 5.1±1.6 pts.). The expression of inflammatory marker genes was not significantly different at 15 minutes and 24 hours after CCI. In contrast, significantly more Iba1-positive cells were present in the pericontusional region after sevo compared to comb anesthesia (sevo = 181±48/mm3; iso = 150±36/mm3; comb = 113±40/mm3). A brief episode of anesthesia, which is sufficient for surgical preparations of mice for procedures such as delivering traumatic brain injury, already has a significant impact on the extent of secondary brain damage.

2-Methoxyestradiol confers neuroprotection and inhibits a maladaptive HIF-1α response after traumatic brain injury in mice.

HIF‐1α is pivotal for cellular homeostasis in response to cerebral ischemia. Pharmacological inhibition of HIF‐1α may reduce secondary brain damage by targeting post‐translational mechanisms associated with its proteasomal degradation and nuclear translocation. This study examined the neuroprotective effects of 2‐methoxyestradiol (2ME2), the involved HIF‐1α‐dependent response, and alternative splicing in exon 14 of HIF‐1α (HIF‐1α∆Ex14) after traumatic brain injury (TBI) in mice. Intraperitoneal 2ME2 administration 30 min after TBI caused a dose‐dependent reduction in secondary brain damage after 24 h. 2ME2 was physiologically tolerated, showed no effects on immune cell brain migration, and mitigated trauma‐induced brain expression of neuropathologically relevant HIF‐1α target genes encoding for Plasminogen activator inhibitor 1 and tumor necrosis factor alpha. Moreover, TBI‐induced expression of pro‐apoptotic BNIP3 was attenuated by 2ME2 treatment. Alternatively, spliced HIF‐1α∆Ex14 was substantially up‐regulated from 6 to 48 h after TBI. In vitro, nuclear location and gene transcription activity of HIF‐1α∆Ex14 were impaired compared to full‐length HIF‐1α, but no effects on nuclear translocation of the transcriptional complex partner HIF‐1β were observed. This study demonstrates that 2ME2 confers neuroprotection after TBI. While the role of alternatively spliced HIF‐1α∆Ex14 remains elusive, the in vivo data provide evidence that inhibition of a maladaptive HIF‐1α‐dependent response contributes to the neuroprotective effects of 2ME2.

Delayed inhibition of angiotensin II receptor type 1 reduces secondary brain damage and improves functional recovery after experimental brain trauma

Objective:To investigate the regulation of the cerebral renin–angiotensin system and the effect of angiotensin II receptor type 1 inhibition on secondary brain damage, cerebral inflammation, and neurologic outcome after head trauma. Design:The expression of renin–angiotensin system components was determined at 15 mins, 3 hrs, 6 hrs, 12 hrs, and 24 hrs after controlled cortical impact in mice. Angiotensin II receptor type 1 was inhibited using candesartan (0.1, 0.5, 1 mg/kg) after trauma to determine its effect on secondary brain damage, brain edema formation, and inflammation. The window of opportunity was tested by delaying angiotensin II receptor type 1 inhibition for 30 mins, 1 hr, 2 hrs, and 4 hrs. The long-term effect was tested by single and daily repeated treatment with candesartan for 5 days after controlled cortical impact. Setting:University research laboratory. Subjects:Male C57Bl/6N mice. Interventions:Brain trauma by use of a controlled cortical impact device. Measurements and Main Results:Expression of angiotensin II receptor type 1A decreased by 42% within 24 hrs after controlled cortical impact, whereas angiotensin II receptor type 1B expression increased to 220% between 6 and 12 hrs. Blockage of angiotensin II receptor type 1with 0.1 mg/kg candesartan within 4 hrs of injury significantly reduced secondary brain damage (30 mins: 25 mm3 vs. vehicle: 41 mm3) and improved neurologic function after 24 hrs but failed to reduce brain edema formation. Daily treatment with candesartan afforded sustained reduction of brain damage and improved neurologic function 5 days after traumatic brain injury compared with single and vehicle treatment. Inhibition of angiotensin II receptor type 1 significantly attenuated posttraumatic inflammation (interleukin-6: −56%; interleukin-1&bgr;: −42%; inducible nitric oxide synthase: −36%; tumor necrosis factor-&agr;: −35%) and microglia activation (vehicle: 163 ± 25/mm2 vs. candesartan: 118 ± 13/mm2). Higher dosages (0.5 and 1 mg/kg) resulted in prolonged reduction in blood pressure and failed to reduce brain lesion. Conclusions:The results indicate that angiotensin II receptor type 1 plays a key role in the development of secondary brain damage after brain trauma. Inhibition of angiotensin II receptor type 1 with a delay of up to 4 hrs after traumatic brain injury effectively reduces lesion volume. This reduction makes angiotensin II receptor type 1 a promising therapeutic target for reducing cerebral inflammation and limiting secondary brain damage.

Xenon Improves Neurologic Outcome and Reduces Secondary Injury Following Trauma in an In Vivo Model of Traumatic Brain Injury

Objectives:To determine the neuroprotective efficacy of the inert gas xenon following traumatic brain injury and to determine whether application of xenon has a clinically relevant therapeutic time window. Design:Controlled animal study. Setting:University research laboratory. Subjects:Male C57BL/6N mice (n = 196). Interventions:Seventy-five percent xenon, 50% xenon, or 30% xenon, with 25% oxygen (balance nitrogen) treatment following mechanical brain lesion by controlled cortical impact. Measurements and Main Results:Outcome following trauma was measured using 1) functional neurologic outcome score, 2) histological measurement of contusion volume, and 3) analysis of locomotor function and gait. Our study shows that xenon treatment improves outcome following traumatic brain injury. Neurologic outcome scores were significantly (p < 0.05) better in xenon-treated groups in the early phase (24 hr) and up to 4 days after injury. Contusion volume was significantly (p < 0.05) reduced in the xenon-treated groups. Xenon treatment significantly (p < 0.05) reduced contusion volume when xenon was given 15 minutes after injury or when treatment was delayed 1 or 3 hours after injury. Neurologic outcome was significantly (p < 0.05) improved when xenon treatment was given 15 minutes or 1 hour after injury. Improvements in locomotor function (p < 0.05) were observed in the xenon-treated group, 1 month after trauma. Conclusions:These results show for the first time that xenon improves neurologic outcome and reduces contusion volume following traumatic brain injury in mice. In this model, xenon application has a therapeutic time window of up to at least 3 hours. These findings support the idea that xenon may be of benefit as a neuroprotective treatment in patients with brain trauma.

Posttraumatic Propofol Neurotoxicity Is Mediated via the Pro-Brain-Derived Neurotrophic Factor-p75 Neurotrophin Receptor Pathway in Adult Mice.

Objectives:The gamma-aminobutyric acid modulator propofol induces neuronal cell death in healthy immature brains by unbalancing neurotrophin homeostasis via p75 neurotrophin receptor signaling. In adulthood, p75 neurotrophin receptor becomes down-regulated and propofol loses its neurotoxic effect. However, acute brain lesions, such as traumatic brain injury, reactivate developmental-like programs and increase p75 neurotrophin receptor expression, probably to foster reparative processes, which in turn could render the brain sensitive to propofol-mediated neurotoxicity. This study investigates the influence of delayed single-bolus propofol applications at the peak of p75 neurotrophin receptor expression after experimental traumatic brain injury in adult mice. Design:Randomized laboratory animal study. Setting:University research laboratory. Subjects:Adult C57BL/6N and nerve growth factor receptor–deficient mice. Interventions:Sedation by IV propofol bolus application delayed after controlled cortical impact injury. Measurements and Main Results:Propofol sedation at 24 hours after traumatic brain injury increased lesion volume, enhanced calpain-induced &agr;II-spectrin cleavage, and increased cell death in perilesional tissue. Thirty-day postinjury motor function determined by CatWalk (Noldus Information Technology, Wageningen, The Netherlands) gait analysis was significantly impaired in propofol-sedated animals. Propofol enhanced pro–brain-derived neurotrophic factor/brain-derived neurotrophic factor ratio, which aggravates p75 neurotrophin receptor–mediated cell death. Propofol toxicity was abolished both by pharmacologic inhibition of the cell death domain of the p75 neurotrophin receptor (TAT-Pep5) and in mice lacking the extracellular neurotrophin binding site of p75 neurotrophin receptor. Conclusions:This study provides first evidence that propofol sedation after acute brain lesions can have a deleterious impact and implicates a role for the pro–brain-derived neurotrophic factor-p75 neurotrophin receptor pathway. This observation is important as sedation with propofol and other compounds with GABA receptor activity are frequently used in patients with acute brain pathologies to facilitate sedation or surgical and interventional procedures.