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

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 mm(3); iso = 20.5±3.7 mm(3); comb = 19.5±4.6 mm(3)). Within 24 hours after injury, lesion size increased in all groups (sevo = 45.3±9.0 mm(3); iso = 31.5±4.0 mm(3); comb = 44.2±6.2 mm(3)). 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/mm(3); iso = 150±36/mm(3); comb = 113±40/mm(3)). 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

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

Angiotensin II receptor 1 blockage limits brain damage and improves functional outcome after brain injury in aged animals despite age-dependent reduction in AT1 expression

Traumatic brain injury (TBI) is a frequent pathology associated with poor neurological outcome in the aged population. We recently observed accelerated cerebral inflammation in aged mice in response to TBI. Candesartan is a potent specific inhibitor of angiotensin II receptor type 1 (AT1) which limits cerebral inflammation and brain damage in juvenile animals after experimental TBI. In the present study, we show significantly lower posttraumatic AT1 mRNA levels in aged (21 months) compared to young (2 months) mice. Despite low cerebral At1 expression, pharmacologic blockade by treatment with candesartan [daily, beginning 30 min after experimental TBI by controlled cortical impact (CCI)] was highly effective in both young and aged animals and reduced histological brain damage by −20% after 5 days. In young mice, neurological improvement was enhanced by AT1 inhibition 5 days after CCI. In older animals, candesartan treatment reduced functional impairment already on day 3 after TBI and post-traumatic body weight (BW) loss was attenuated. Candesartan reduced microglia activation (−40%) in young and aged animals, and neutrophil infiltration (−40% to 50%) in aged mice, whereas T-cell infiltration was not changed in either age group. In young animals, markers of anti-inflammatory microglia M2a polarization [arginase 1 (Arg1), chitinase3-like 3 (Ym1)] were increased by candesartan at days 1 and 5 after insult. In older mice 5 days after insult, expression of Arg1 was significantly higher independently of the treatment, whereas Ym1 gene expression was further enhanced by AT1 inhibition. Despite age-dependent posttraumatic differences in At1 expression levels, inhibition of AT1 was highly effective in a posttreatment paradigm. Targeting inflammation with candesartan is, therefore, a promising therapeutic strategy to limit secondary brain damage independent of the age

Expression of inflammatory marker genes and microglia activation.

<p><b>A–D:</b> The mRNA expression was determined in contused brain tissue. Expression of the inflammatory marker genes COX-2 (<b>A</b>), iNOS (<b>B</b>), and IL6 (<b>C</b>) was not significantly different between the anesthetic protocols at 15 minutes and 24 hours after experimental brain trauma (group size: naïve, n = 9; 15 minutes, n = 6; 24 hours, n = 8; data are presented as mean ± S.D.). <b>D:</b> As a marker for activated microglia, immunohistochemical analysis of Iba-1-positive cells was performed in the pericontusional tissue that revealed a significantly increased activation in sevo animals vs. comb (n = 8 per group; sevo = sevoflurane; iso = isoflurane; comb = i.p. injection of midazolam, fentanyl and medetomidine; native = no surgery; NS = not significant; p-values were adjusted for multiple comparison by Bonferroni). <b>E:</b> Example pictures of the Iba-1-stained slides of animals 24 hours post-injury (20× magnification). The number of activated microglia was determined at the border zone adjacent to the damaged brain tissue. Iba-1-positive cells are labeled dark brown (arrows).</p

Histological brain damage and neurological function.

<p>(<b>A</b>) The contusion volume was determined 15 minutes and 24 hours after experimental brain trauma (CCI). The primary lesion 15 minutes after CCI was not significantly different between the anesthetic regimens. Within 24 hours, brain contusions increased significantly in all groups. Sevo-anesthetized animals experienced a significantly larger contusion volume compared to the isoflurane and comb groups (group size: 15 minutes, n = 6 per group; 24 hours, n = 8). (<b>B</b>) The contusion areas of all groups obtained from 11 coronal brain sections (750-µm interval) cut from rostral (no. 1) to dorsal (no. 11) 15 minutes and 24 hours after CCI. The graph illustrates that the secondary expansion of brain damage occurred in all brain areas (mean ± S.E.M.). (<b>C</b>) The body weight was determined 24 hours after CCI and was compared to the value obtained before trauma as the general marker for the well-being of the animals. Animals anesthetized with comb lost significantly more body weight compared to iso. The other anesthetic regimens were not significantly different. (<b>D</b>) Neurological function was determined by the neurological severity score (NSS; 0 point = no impairment; 10 points = maximal impairment, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019948#pone-0019948-t002" target="_blank"><b>Table 2</b></a>) 23 hours after CCI. NNS scores of comb were significantly worse compared to iso. The other groups were not significantly different. (sevo = sevoflurane; iso = isoflurane; comb = i.p. injection of midazolam, fentanyl and medetomidine; data are presented as mean ± S.D.; NS = not significant; p-values are adjusted for multiple comparison by Bonferroni).</p

Criteria of the Neurological Severity Score (NSS).

<p>One point is awarded for failure to perform a task.</p