Inhaled Nitric Oxide Protects Cerebral Autoregulation and Reduces Hippocampal Necrosis After Traumatic Brain Injury Through Inhibition of ET-1, ERK MAPK and IL-6 Upregulation in Pigs.

OBJECTIVE: Traumatic brain injury (TBI) is an important contributor to morbidity and mortality. Cerebral autoregulation is impaired after TBI, contributing to poor outcome. Extracellular signal-related kinase (ERK) mitogen activated protein kinase (MAPK) and ET-1 are upregulated and contribute to impairment of cerebral autoregulation and histopathology after porcine fluid percussion brain injury (FPI). Recent studies show that inhaled nitric oxide (iNO) prevents impairment of cerebral autoregulation and histopathology after FPI in pigs. Unrelated studies indicated an association between ERK and increased IL-6 after FPI. However, the role of IL-6 in central nervous system (CNS) pathology is not well understood. We investigated whether iNO protects autoregulation and limits histopathology after FPI in pigs due to modulation of brain injury associated upregulation of ET-1, ERK MAPK, and IL-6. METHODS: Lateral FPI was produced in anesthetized pigs equipped with a closed cranial window and iNO administered at 30 min or 2 h post injury. RESULTS: CSF ET-1, ERK MAPK, and IL-6 were increased by FPI, but release was blocked by iNO administered at 30 min or 2 h after TBI. The IL-6 antagonist LMT-28 prevented impairment of cerebral autoregulation and hippocampal CA1 and CA3 neuronal necrosis after FPI. Papaverine induced dilation was unchanged by FPI and LMT-28. Protection lasted for at least 2 h after iNO administration was stopped. CONCLUSIONS: These data indicate that iNO protects cerebral autoregulation and reduces hippocampal necrosis after traumatic brain injury through inhibition of ET-1, ERK MAPK, and IL-6 upregulation in pigs.

Sex and age differences in phenylephrine mechanisms and outcomes after piglet brain injury.

BackgroundTraumatic brain injury (TBI) is the leading cause of injury-related death in children, with boys and children under 4 years of age having particularly poor outcomes. Cerebral autoregulation is often impaired after TBI, contributing to poor outcome. In prior studies on newborn pigs, phenylephrine (Phe) preferentially protected cerebral autoregulation in female but not in male subjects after TBI. We hypothesized that, in contrast to the newborn, Phe prevents impairment of autoregulation and tissue injury following TBI in both sexes of older pigs.MethodsCerebral autoregulation, cerebrospinal fluid (CSF) extracellular signal-related kinase (ERK) and endothelin, and histopathology were determined after moderate fluid percussion brain injury (FPI) in male and female juvenile pigs after Phe.ResultsAutoregulation was more impaired in male than in female subjects. Phe protects autoregulation in both sexes after FPI, blocks ERK and endothelin, and decreases the number of necrotic neurons in male and female subjects after FPI.ConclusionsThese data indicate that Phe protects autoregulation and limits neuronal necrosis via blockage of ERK and endothelin after FPI in male and female subjects. Together with prior observations in newborn pigs where Phe protected autoregulation in female but not in male subjects, these data suggest that use of Phe to improve outcomes after TBI is both sex- and age-dependent.

Microparticles Impair Hypotensive Cerebrovasodilation and Cause Hippocampal Neuronal Cell Injury after Traumatic Brain Injury.

Endothelin-1 (ET-1), tissue plasminogen activator (tPA), and extracellular signal-regulated kinases-mitogen activated protein kinase (ERK-MAPK) are mediators of impaired cerebral hemodynamics after fluid percussion brain injury (FPI) in piglets. Microparticles (MPs) are released into the circulation from a variety of cells during stress, are pro-thrombotic and pro-inflammatory, and may be lysed with polyethylene glycol telomere B (PEG-TB). We hypothesized that MPs released after traumatic brain injury impair hypotensive cerebrovasodilation and that PEG-TB protects the vascular response via MP lysis, and we investigated the relationship between MPs, tPA, ET-1, and ERK-MAPK in that process. FPI was induced in piglets equipped with a closed cranial window. Animals received PEG-TB or saline (vehicle) 30-minutes post-injury. Serum and cerebrospinal fluid (CSF) were sampled and pial arteries were measured pre- and post-injury. MPs were quantified by flow cytometry. CSF samples were analyzed with enzyme-linked immunosorbent assay. MP levels, vasodilatory responses, and CSF signaling assays were similar in all animals prior to injury and treatment. After injury, MP levels were elevated in the serum of vehicle but not in PEG-TB-treated animals. Pial artery dilation in response to hypotension was impaired after injury but protected in PEG-TB-treated animals. After injury, CSF levels of tPA, ET-1, and ERK-MAPK were all elevated, but not in PEG-TB-treated animals. PEG-TB-treated animals also showed reduction in neuronal injury in CA1 and CA3 hippocampus, compared with control animals. These results show that serum MP levels are elevated after FPI and lead to impaired hypotensive cerebrovasodilation via over-expression of tPA, ET-1, and ERK-MAPK. Treatment with PEG-TB after injury reduces MP levels and protects hypotensive cerebrovasodilation and limits hippocampal neuronal cell injury.

Salvinorin A administration after global cerebral hypoxia/ischemia preserves cerebrovascular autoregulation via kappa opioid receptor in piglets.

BACKGROUND: Cerebral hypoxia/ischemia (HI) is not uncommon during the perinatal period. If occurring, it can result in severe neurologic disabilities that persist throughout life. Salvinorin A, a non-opioid Kappa opioid receptors (KOR) selective agonist, has the potential to address this devastating situation. We have demonstrated that salvinorin A administration before HI, preserves pial artery autoregulative function through both the KOR and extracellular signal-regulated kinases (ERK) pathways. In the present study, we tested the hypothesis that administration of salvinorin A after HI could preserve cerebral autoregulation via KOR and ERK pathway. METHODOLOGY/PRINCIPAL FINDINGS: The response of the pial artery to hypercapnia, hypotension and isoproterenol were monitored before and 1 hour after HI in piglets equipped with a cranial window. Four groups of drug administration were performed after HI. The control group had DMSO (1 µl/kg, i.v.) administrated immediately after HI. Two salvinorin A treated groups had salvinorin A (10 µg/kg, i.v.) administrated 0 and 30 min after HI, respectively. The 4(th) group had salvinorin A and the KOR antagonist norbinaltorphimine (Nor-BIN, 1 µM topical) co-administrated 0 min after HI (n = 5). The dilation responses of the pial artery to hypercapnia and hypotension were impaired after global HI and were preserved with salvinorin A administration immediately or 30 min after HI. The preservation of autoregulation was abolished when nor-BIN was administered. Levels of phosphor-ERK(pERK)/ERK in the cerebrospinal fluid (CSF) were measured before and 1 hour after HI. After HI, the pERK/ERK levels significantly increased in both DMSO control group and salvinorin A and nor-BIN co-administration group. The elevated levels of pERK/ERK were not observed with salvinorin A only groups. CONCLUSIONS: Salvinorin A administration 0 and 30 min after HI preserves autoregulation of pial artery to hypercapnia and hypotension via kappa opioid receptor and ERK pathway.

Salvinorin A pretreatment preserves cerebrovascular autoregulation after brain hypoxic/ischemic injury via extracellular signal-regulated kinase/mitogen-activated protein kinase in piglets.

BACKGROUND: Cerebral hypoxia/ischemia during infant congenital heart surgery is not uncommon and may induce devastating neurologic disabilities persistent over the lifespan. Hypoxia/ischemia-induced cerebrovascular dysfunction is thought to be an important contributor to neurological damage. No pharmacological agents have been found to prevent this. Mitogen activated protein kinase (MAPK), including extracellular signal regulated kinase (ERK), c-Jun-N-terminal kinase, and p38, is thought to contribute to ischemic preconditioning. We investigated whether pretreatment with salvinorin A, the only natural nonopioid κ receptor agonist, could preserve autoregulation of the pial artery via MAPK. METHODS: The response of the pial artery to hypotension and hypercapnia was monitored in piglets equipped with a closed cranial window before and after hypoxia and ischemia in the presence or absence of U0126, an inhibitor for the protein kinase upstream of ERK, sp600125, an inhibitor of c-Jun-N-terminal kinase or sb203580, an inhibitor of p38. Salvinorin A (10 µg/kg IV) was administered 30 minutes before hypoxia/ischemia in salvinorin-treated animals. Cerebrospinal fluid samples were collected before and 30 minutes after salvinorin A administration for the measurement of MAPK. Data (n = 5) were analyzed by repeated-measures analysis of variance. RESULTS: Pial artery dilation to hypercapnia and hypotension was blunted after hypoxia/ ischemia but preserved well by pretreatment with salvinorin A. U0126, but not sp600125 or sb203580, abolished the preservative effects of salvinorin A on cerebral vascular autoregulation to hypotension and hypercapnia. The ratio of pERK/ERK in cerebrospinal fluid increased significantly in salvinorin-treated animals, which was inhibited by U0126. CONCLUSIONS: Salvinorin A pretreatment preserves autoregulation of the pial artery to hypotension and hypercapnia after hypoxia/ischemia via ERK in a piglet model.

SNP improves cerebral hemodynamics during normotension but fails to prevent sex dependent impaired cerebral autoregulation during hypotension after brain injury.

Traumatic brain injury (TBI) is a leading cause of morbidity in children and boys are disproportionately represented. Hypotension is common and worsens outcome after TBI. Previous studies show that adrenomedullin, a cerebrovasodilator, prevented sex dependent impairment of autoregulation during hypotension after piglet fluid percussion brain injury (FPI). We hypothesized that this concept was generalizable and that administration of another vasodilator, sodium nitroprusside (SNP), may equally improve CBF and cerebral autoregulation in a sex dependent manner after FPI. SNP produced equivalent percent cerebrovasodilation in male and female piglets. Reductions in pial artery diameter, cortical CBF, and cerebral perfusion pressure (CPP) concomitant with elevated intracranial pressure (ICP) after FPI were greater in male compared to female piglets during normotension which was blunted by SNP. During hypotension, pial artery dilation (PAD) was impaired more in the male than the female after FPI. However, SNP did not improve hypotensive PAD after FPI in females and paradoxically caused vasoconstriction in males. SNP did not prevent reductions in CBF, CPP or autoregulatory index during combined hypotension and FPI in either sex. SNP aggravated ERK MAPK upregulation after FPI. These data indicate that despite prevention of reductions in CBF after FPI, SNP does not prevent impairment of autoregulation during hypotension after FPI. These data suggest that therapies directed at a purely hemodynamic increase in CPP will fail to improve outcome during combined TBI and hypotension.

Adrenomedullin prevents sex-dependent impairment of autoregulation during hypotension after piglet brain injury through inhibition of ERK MAPK upregulation.

Cerebrospinal fluid (CSF) adrenomedullin (ADM) levels are increased in female, but remain unchanged in male, piglets after fluid percussion injury (FPI) of the brain. Subthreshold vascular concentrations of ADM restore impaired hypotensive pial artery dilation after FPI more in males than females. Extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK) is upregulated and contributes to reductions in cerebral blood flow (CBF) after FPI. We hypothesized that ADM prevents sex-dependent impairment of autoregulation during hypotension after FPI through inhibition of ERK MAPK upregulation. FPI increased ERK MAPK more in males than in females. CBF was unchanged during hypotension in sham animals, was reduced more in males than in females after FPI during normotension, and was further reduced in males than in females during hypotension and after FPI. ADM and the ERK MAPK antagonist U 0126 prevented reductions in CBF during hypotension and FPI more in males than in females. Transcranial Doppler (TCD) blood flow velocity was unchanged during hypotension in sham animals, was decreased during hypotension and FPI in male but not in female pigs, and was ameliorated by ADM. Intracranial pressure (ICP) was increased after FPI more in male than in female animals. ADM blunted elevated ICP during FPI and hypotension in males, but not in females. ADM prevented reductions in cerebral perfusion pressure (CPP) during FPI and hypotension in males but not in females. The calculated autoregulatory index was unchanged during hypotension in sham animals, but was reduced more in males than females during hypotension and FPI. ADM prevented reductions in autoregulation during hypotension and FPI more in males than females. These data indicate that ADM prevented loss of cerebral autoregulation after FPI in a sex-dependent and ERK MAPK-dependent manner.

Measurement of ERK 1/2 in CSF from patients with neuropsychiatric disorders and evidence for the presence of the activated form.

The clinical diagnosis of neurodegenerative disorders can be supported by soluble biomarkers in cerebrospinal fluid (CSF), such as tau protein, phospho-tau, and amyloid-beta peptides. In particular, increased CSF levels of phospho-tau in Alzheimer's disease appear to reflect disease specific pathological processes. We report here evidence for the presence of soluble MAP-kinase ERK1/2 in a small set of human CSF samples from patients with Alzheimer's disease, frontotemporal degeneration, and mild cognitive impairment. The level of total ERK1/2 in CSF as measured by electrochemiluminescent assay was correlated with that of total tau and phospho-tau. A small fraction of ERK1/2 in a pooled CSF sample was found to be in the doubly phosphorylated (activated) state. Our findings suggest that i) MAP kinase ERK1/2 is apparently released under neurodegenerative conditions in parallel with tau and phospho-tau and ii) in the future, it might be possible to find in CSF samples evidence for disease related alterations in brain kinase signaling pathways by use of highly sensitive and activation-state specific anti-kinase antibodies.

Red blood cells-coupled tPA prevents impairment of cerebral vasodilatory responses and tissue injury in pediatric cerebral hypoxia/ischemia through inhibition of ERK MAPK activation.

Babies experience hypoxia (H) and ischemia (I) from stroke. The only approved treatment for stroke is fibrinolytic therapy with tissue-type plasminogen activator (tPA). However, tPA potentiates H/I-induced impairment of responses to cerebrovasodilators such as hypercapnia and hypotension, and blockade of tPA-mediated vasoactivity prevents this deleterious effect. Coupling of tPA to red blood cells (RBCs) reduces its central nervous system (CNS) toxicity through spatially confining the drug to the vasculature. Mitogen-activated protein kinase (MAPK), a family of at least three kinases, is upregulated after H/I. In this study we determined whether RBC-tPA given before or after cerebral H/I would preserve responses to cerebrovasodilators and prevent neuronal injury mediated through the extracellular signal-related kinase (ERK) MAPK pathway. Animals given RBC-tPA maintained responses to cerebrovasodilators at levels equivalent to pre-H/I values. cerebrospinal fluid and brain parenchymal ERK MAPK was elevated by H/I and this upregulation was potentiated by tPA, but blunted by RBC-tPA. U0126, an ERK MAPK antagonist, also maintained cerebrovasodilation post H/I. Neuronal degeneration in CA1 hippocampus after H/I was not improved by tPA, but was ameliorated by RBC-tPA and U0126. These data suggest that coupling of tPA to RBCs offers a novel approach toward increasing the benefit/risk ratio of thrombolytic therapy for CNS disorders associated with H/I.