RESUMO
Kynurenic acid (KYNA), an endogenous neuroprotectant with antiexcitotoxic, antioxidant, and anti-inflammatory effects, is synthesized through the tryptophan-kynurenine (KYN) pathway. We investigated whether brain KYN or KYNA levels were affected by asphyxia in a translational piglet model of hypoxic-ischemic encephalopathy (HIE). We also studied brain levels of the putative blood-brain barrier (BBB) permeable neuroprotective KYNA analogue SZR72, and whether SZR72 or therapeutic hypothermia (TH) modified KYN or KYNA levels. KYN, KYNA, and SZR72 levels were determined using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry in five brain regions 24 h after 20 min of asphyxia in vehicle-, SZR72- and TH-treated newborn piglets (n = 6-6-6) and naive controls (n = 4). Endogenous brain KYN levels (median range 311.2-965.6 pmol/g) exceeded KYNA concentrations (4.5-6.0 pmol/g) ~100-fold. Asphyxia significantly increased cerebral KYN and KYNA levels in all regions (1512.0-3273.9 and 16.9-21.2 pmol/g, respectively), increasing the KYN/Tryptophan-, but retaining the KYNA/KYN ratio. SZR72 treatment resulted in very high cerebral SZR72 levels (13.2-33.2 nmol/g); however, KYN and KYNA levels remained similar to those of the vehicle-treated animals. However, TH virtually ameliorated asphyxia-induced elevations in brain KYN and KYNA levels. The present study reports for the first time that the KYN pathway is altered during HIE development in the piglet. SZR72 readily crosses the BBB in piglets but fails to affect cerebral KYNA levels. Beneficial effects of TH may include restoration of the tryptophan metabolism to pre-asphyxia levels.
Assuntos
Hipotermia , Hipóxia-Isquemia Encefálica , Suínos , Animais , Cinurenina/metabolismo , Triptofano/metabolismo , Ácido Cinurênico/metabolismo , Asfixia , Hipóxia-Isquemia Encefálica/terapiaRESUMO
Hypoxic-ischemic encephalopathy (HIE) remains to be a major cause of long-term neurodevelopmental deficits in term neonates. Hypothermia offers partial neuroprotection warranting research for additional therapies. Kynurenic acid (KYNA), an endogenous product of tryptophan metabolism, was previously shown to be beneficial in rat HIE models. We sought to determine if the KYNA analog SZR72 would afford neuroprotection in piglets. After severe asphyxia (pHa = 6.83 ± 0.02, ΔBE = -17.6 ± 1.2 mmol/L, mean ± SEM), anesthetized piglets were assigned to vehicle-treated (VEH), SZR72-treated (SZR72), or hypothermia-treated (HT) groups (n = 6, 6, 6; Tcore = 38.5, 38.5, 33.5 °C, respectively). Compared to VEH, serum KYNA levels were elevated, recovery of EEG was faster, and EEG power spectral density values were higher at 24 h in the SZR72 group. However, instantaneous entropy indicating EEG signal complexity, depression of the visual evoked potential (VEP), and the significant neuronal damage observed in the neocortex, the putamen, and the CA1 hippocampal field were similar in these groups. In the caudate nucleus and the CA3 hippocampal field, neuronal damage was even more severe in the SZR72 group. The HT group showed the best preservation of EEG complexity, VEP, and neuronal integrity in all examined brain regions. In summary, SZR72 appears to enhance neuronal activity after asphyxia but does not ameliorate early neuronal damage in this HIE model.
Assuntos
Asfixia Neonatal/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Ácido Cinurênico/análogos & derivados , Neurônios/metabolismo , Animais , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Região CA1 Hipocampal/diagnóstico por imagem , Região CA1 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/diagnóstico por imagem , Região CA3 Hipocampal/efeitos dos fármacos , Modelos Animais de Doenças , Eletroencefalografia , Potenciais Evocados Visuais/efeitos dos fármacos , Humanos , Ácido Cinurênico/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Pesquisa Translacional BiomédicaRESUMO
Hypoxic-ischemic encephalopathy (HIE) is still a major cause of neonatal death and disability as therapeutic hypothermia (TH) alone cannot afford sufficient neuroprotection. The present study investigated whether ventilation with molecular hydrogen (2.1% H2) or graded restoration of normocapnia with CO2 for 4 h after asphyxia would augment the neuroprotective effect of TH in a subacute (48 h) HIE piglet model. Piglets were randomized to untreated naïve, control-normothermia, asphyxia-normothermia (20-min 4%O2-20%CO2 ventilation; Tcore = 38.5 °C), asphyxia-hypothermia (A-HT, Tcore = 33.5 °C, 2-36 h post-asphyxia), A-HT + H2, or A-HT + CO2 treatment groups. Asphyxia elicited severe hypoxia (pO2 = 19 ± 5 mmHg) and mixed acidosis (pH = 6.79 ± 0.10). HIE development was confirmed by altered cerebral electrical activity and neuropathology. TH was significantly neuroprotective in the caudate nucleus but demonstrated virtually no such effect in the hippocampus. The mRNA levels of apoptosis-inducing factor and caspase-3 showed a ~10-fold increase in the A-HT group compared to naïve animals in the hippocampus but not in the caudate nucleus coinciding with the region-specific neuroprotective effect of TH. H2 or CO2 did not augment TH-induced neuroprotection in any brain areas; rather, CO2 even abolished the neuroprotective effect of TH in the caudate nucleus. In conclusion, the present findings do not support the use of these medical gases to supplement TH in HIE management.
Assuntos
Asfixia Neonatal/terapia , Dano Encefálico Crônico/prevenção & controle , Dióxido de Carbono/uso terapêutico , Hidrogênio/uso terapêutico , Hipotermia Induzida , Hipóxia-Isquemia Encefálica/terapia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Acidose/sangue , Acidose/etiologia , Acidose/prevenção & controle , Administração por Inalação , Animais , Animais Recém-Nascidos , Fator de Indução de Apoptose/biossíntese , Fator de Indução de Apoptose/genética , Asfixia Neonatal/complicações , Asfixia Neonatal/tratamento farmacológico , Dano Encefálico Crônico/etiologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Dióxido de Carbono/administração & dosagem , Dióxido de Carbono/toxicidade , Caspase 3/biossíntese , Caspase 3/genética , Núcleo Caudado/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eletroencefalografia , Potenciais Evocados Visuais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/patologia , Hidrogênio/administração & dosagem , Hidrogênio/análise , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/patologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/administração & dosagem , Especificidade de Órgãos , Distribuição Aleatória , SuínosRESUMO
Cyclooxygenase-2 (COX-2) has an established role in the pathogenesis of hypoxic-ischemic encephalopathy (HIE). In this study we sought to determine whether COX-2 was induced by asphyxia in newborn pigs, and whether neuronal COX-2 levels were affected by H2 treatment. Piglets were subjected to either 8 min of asphyxia or a more severe 20 min of asphyxia followed by H2 treatment (inhaling room air containing 2.1% H2 for 4 h). COX-2 immunohistochemistry was performed on brain samples from surviving piglets 24 h after asphyxia. The percentages of COX-2-immunopositive neurons were determined in cortical and subcortical areas. Only in piglets with more severe HIE, we observed significant, region-specific increases in neuronal COX-2 expression within the parietal and occipital cortices and in the CA3 hippocampal subfield. H2 treatment essentially prevented the increases in COX-2-immunopositive neurons. In the parietal cortex, the attenuation of COX-2 induction was associated with reduced 8'-hydroxy-2'-deoxyguanozine immunoreactivity and retained microglial ramifcation index, which are markers of oxidative stress and neuroinfiammation, respectively. This study demonstrates for the first time that asphyxia elevates neuronal COX-2 expression in a piglet HIE model. Neuronal COX-2 induction may play region-specific roles in brain lesion progression during HIE development, and inhibition of this response may contribute to the antioxidant/anti-infiammatory neuroprotective effects of H2 treatment.
Assuntos
Asfixia/prevenção & controle , Ciclo-Oxigenase 2/metabolismo , Hidrogênio/uso terapêutico , Hipóxia-Isquemia Encefálica/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Animais , Animais Recém-Nascidos , Hipocampo/fisiopatologia , Masculino , Microglia/metabolismo , Neurônios/metabolismo , Lobo Parietal/fisiopatologia , SuínosRESUMO
Brain interstitial pH (pHbrain) alterations play a crucial role in the development of hypoxic-ischemic (HI) encephalopathy (HIE) caused by asphyxia in neonates. The newborn pig is one of the most suitable large animal models for studying HIE, however, compared to rats, experimental data on pHbrain alterations during HIE induction are limited. The major objective of the present study was thus to compare pHbrain changes during HIE development induced by experimental normocapnic hypoxia (H) or asphyxia (A), elicited with ventilation of a gas mixture containing 6%O2 or 6%O2/20%CO2, respectively for 20 min, under either normothermia (NT) or hypothermia (HT) (38.5 ± 0.5 °C or 33.5 ± 0.5 °C core temperature, respectively) in anesthetized piglets yielding four groups: H-NT, A-NT, H-HT, and A-HT. pHbrain changes during HI stress and the 60 min reoxygenation period were measured using a pH-selective microelectrode inserted into the parietal cortex through an open cranial window. In all groups, the pHbrain response to HI stress was acidosis, at the nadir pHbrain values dropped from the baseline of 7.27 ± 0.02 to H-NT:5.93 ± 0.30, A-NT:5.90 ± 0.52, H-HT:6.81 ± 0.27, and A-HT:6.27 ± 0.24 indicating that (1) H and A elicited similar, severe brain acidosis under NT greatly exceeding pH changes in arterial blood (pHa dropped to 7.24 ± 0.07 and 6.78 ± 0.03 from 7.52 ± 0.06 and 7.50 ± 0.05, respectively), and (2) HT ameliorated more the brain acidosis induced by H than by A. In all four groups, pHbrain was restored to baseline values without an alkalotic overshoot during the observed reoxygenation, Our findings suggest that under NT either H or A - both commonly employed HI stresses to elicit HIE in piglet models - would result in a similar acidotic pHbrain response without an alkalotic component either during the HI stress or the early reoxygenation period.
RESUMO
A new laser speckle-contrast analysis (LASCA) technique based on multi-exposure imaging was employed to simultaneously study pial arteriolar responses with cerebrocortical perfusion changes to various vasodilator (5-10% CO(2) ventilation, bradykinin (1-10 µM), N-methyl-D-aspartate (100 µM)) vasoconstrictor (10-100 µM noradrenaline, 1M KCl), or neutral (2.1% H(2) ventilation) stimuli as well as to asphyxia in the newborn piglet. Anesthetized, ventilated animals (n=20) were fitted with closed cranial windows. Multiple exposure laser-speckle image series (1-100 ms) were obtained using a near infrared diode laser (λ=808 nm). The autocorrelation decay time (τ) of speckle fluctuations was determined over pial arterioles and parenchymal areas to express 1/τ being proportional to blood flow velocity by two different LASCA techniques: our novel multi-exposure or a single exposure (2 and 20 ms) approach. 1/τ values yielded by different LASCA techniques were not significantly different at most points. LASCA easily detected both increases and decreases in cortical blood flow (CoBF). Cortical 1/τ changes to hypercapnia closely matched quantitative CoBF data determined previously, and were also in accordance with increases of pial arteriolar blood flow, calculated from arteriolar flow velocity and cross sectional area changes. In summary, LASCA emerges as an appealing method to simultaneously study microvascular reactivity and cortical perfusion changes in the piglet.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microcirculação , Algoritmos , Animais , Animais Recém-Nascidos , Arteríolas/fisiologia , Velocidade do Fluxo Sanguíneo/fisiologia , Bradicinina/farmacologia , Circulação Cerebrovascular , Meios de Contraste/farmacologia , Relação Dose-Resposta a Droga , Lasers , Modelos Estatísticos , N-Metilaspartato/farmacologia , Pia-Máter/irrigação sanguínea , Suínos , Fatores de Tempo , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
Cyclooxygenase (COX)-2 is the major constitutively expressed COX isoform in the newborn brain. COX-2 derived prostanoids and reactive oxygen species appear to play a major role in the mechanism of perinatal hypoxic-ischemic injury in the newborn piglet, an accepted animal model of the human term neonate. The study aimed to quantitatively determine COX-2 immunopositive neurons in different brain regions in piglets under normoxic conditions (n=15), and 4 hours after 10 min asphyxia (n=11). Asphyxia did not induce significant changes in neuronal COX-2 expression of any studied brain areas. In contrast, there was a marked regional difference in all experimental groups. Thus, significant difference was observed between fronto-parietal and temporo-occipital regions: 59±4% and 67±3% versus 41±2%* and 31±3%* respectively (mean±SEM, data are pooled from all subjects, n=26, *p<0.05, vs. fronto-parietal region). In the hippocampus, COX-2 immunopositivity was rare (highest expression in CA1 region: 14±2%). The studied subcortical areas showed negligible COX-2 staining. Our findings suggest that asphyxia does not significantly alter the pattern of neuronal COX-2 expression in the early reventilation period. Furthermore, based on the striking differences observed in cortical neuronal COX-2 distribution, the contribution of COX-2 mediated neuronal injury after asphyxia may also show region-specific differences.
RESUMO
Hydrogen (H2) has been reported to neutralize toxic reactive oxygen species. Oxidative stress is an important mechanism of neuronal damage after perinatal asphyxia. We examined whether 2.1% H2-supplemented room air (H2-RA) ventilation would preserve cerebrovascular reactivity (CR) and brain morphology after asphyxia/reventilation (A/R) in newborn pigs. Anesthetized, ventilated piglets were assigned to one of the following groups: A/R with RA or H2-RA ventilation (A/R-RA and A/R-H2-RA; n = 8 and 7, respectively) and respective time control groups (n = 9 and 7). Asphyxia was induced by suspending ventilation for 10 min, followed by reventilation with the respective gases for 4 h. After euthanasia, the brains were processed for neuropathological examination. Pial arteriolar diameter changes to graded hypercapnia (5-10% CO2 inhalation), and NMDA (10(-4) M) were determined using the closed cranial window/intravital microscopy before and 1 h after asphyxia. Neuropathology revealed that H2-RA ventilation significantly reduced neuronal injury induced by A/R in virtually all examined brain regions including the cerebral cortex, the hippocampus, basal ganglia, cerebellum, and the brainstem. Furthermore, H2-RA ventilation significantly increased CR to hypercapnia after A/R (% vasodilation was 23 ± 4% versus 41 ± 9%, p < 0.05). H2-RA ventilation did not affect reactive oxygen species-dependent CR to NMDA. In summary, H2-RA could be a promising approach to reduce the neurologic deficits after perinatal asphyxia.
Assuntos
Asfixia Neonatal/fisiopatologia , Encéfalo , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Hidrogênio/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Animais Recém-Nascidos , Análise Química do Sangue , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Hemodinâmica , Humanos , Hipercapnia/metabolismo , Recém-Nascido , SuínosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0233851.].
RESUMO
Brain interstitial pH (pHbrain) alterations play an important role in the mechanisms of neuronal injury in neonatal hypoxic-ischemic encephalopathy (HIE) induced by perinatal asphyxia. The newborn pig is an established large animal model to study HIE, however, only limited information on pHbrain alterations is available in this species and it is restricted to experimental perinatal asphyxia (PA) and the immediate reventilation. Therefore, we sought to determine pHbrain over the first 24h of HIE development in piglets. Anaesthetized, ventilated newborn pigs (n = 16) were instrumented to control major physiological parameters. pHbrain was determined in the parietal cortex using a pH-selective microelectrode. PA was induced by ventilation with a gas mixture containing 6%O2-20%CO2 for 20 min, followed by reventilation with air for 24h, then the brains were processed for histopathology assessment. The core temperature was maintained unchanged during PA (38.4±0.1 vs 38.3±0.1°C, at baseline versus the end of PA, respectively; mean±SEM). In the arterial blood, PA resulted in severe hypoxia (PaO2: 65±4 vs 23±1*mmHg, *p<0.05) as well as acidosis (pHa: 7.53±0.03 vs 6.79±0.02*) that is consistent with the observed hypercapnia (PaCO2: 37±3 vs 160±6*mmHg) and lactacidemia (1.6±0.3 vs 10.3±0.7*mmol/L). Meanwhile, pHbrain decreased progressively from 7.21±0.03 to 5.94±0.11*. Reventilation restored pHa, blood gases and metabolites within 4 hours except for PaCO2 that remained slightly elevated. pHbrain returned to 7.0 in 29.4±5.5 min and then recovered to its baseline level without showing secondary alterations during the 24 h observation period. Neuropathological assessment also confirmed neuronal injury. In conclusion, in spite of the severe acidosis and alterations in blood gases during experimental PA, pHbrain recovered rapidly and notably, there was no post-asphyxia hypocapnia that is commonly observed in many HIE babies. Thus, the neuronal injury in our piglet model is not associated with abnormal pHbrain or low PaCO2 over the first 24 h after PA.
Assuntos
Encéfalo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Acidose/sangue , Acidose/complicações , Acidose/metabolismo , Acidose/fisiopatologia , Animais , Animais Recém-Nascidos , Asfixia Neonatal/sangue , Asfixia Neonatal/metabolismo , Asfixia Neonatal/fisiopatologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Hemodinâmica , Concentração de Íons de Hidrogênio , Hipercapnia/sangue , Hipercapnia/complicações , Hipercapnia/metabolismo , Hipercapnia/fisiopatologia , Hipóxia-Isquemia Encefálica/sangue , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/fisiopatologia , Masculino , Neurônios/patologia , Oxigênio/metabolismo , SuínosRESUMO
Cerebral ischemia/reperfusion (I/R) results in cellular energy failure and dysfunction of the neurovascular unit that contribute to subsequent neuronal cell death in the neonate. PX-18 is a putative neuroprotective inhibitor of secretory phospholipase A(2) (sPLA(2)) but its in vivo testing has been limited by its poor solubility. Our purpose was to assess whether PX-18 preserved neuronal-vascular reactivity to I/R-sensitive endothelium-dependent (hypercapnia, bradykinin) and/or neuron-dependent (N-methyl-D-aspartate; NMDA) stimuli. To make the drug available for in vivo studies, PX-18 was formulated as a 3% nanosuspension applying high pressure homogenization. Newborn piglets (1-day old, n=40) were anesthetized and ventilated, and cerebrovascular reactivity to the above stimuli was determined by measuring changes in pial arteriolar diameters using the closed cranial window/intravital videomicroscopy technique. Intravenous infusion of PX-18 nanosuspension (6 mg/kg, 20 min) did not affect baseline arteriolar diameters, or hypercapnia-, bradykinin-, or NMDA-induced pial arteriolar vasodilation under normoxic conditions. Global cerebral ischemia (10 min) followed by 1 h of reperfusion significantly attenuated hypercapnia-, bradykinin-, and NMDA-induced vasodilation in untreated or vehicle-treated controls. However, PX-18 resulted in nearly full preservation of cerebrovascular reactivity to all these stimuli. In conclusion, inhibition of sPLA(2) by PX-18 improves neurovascular function both at the neuronal and the microvascular level following I/R. This effect of PX-18 likely contributes to its neuroprotective effect.
Assuntos
Ácidos Alcanossulfônicos/farmacologia , Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Oleicos/farmacologia , Fosfolipases A2 Secretórias/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Bradicinina/farmacologia , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/inervação , Química Farmacêutica/métodos , Avaliação Pré-Clínica de Medicamentos , Hipercapnia/fisiopatologia , Microscopia de Vídeo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Tamanho da Partícula , Pia-Máter/irrigação sanguínea , Pia-Máter/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Suínos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologiaRESUMO
Gram-negative bacterial endotoxin lipopolysaccharide (LPS) administration has been used as an animal model of sepsis-related acute lung injury and adult respiratory distress syndrome (ALI/ARDS). This paper describes the lung histology following lung injury induced by the intraperitoneal (i.p.) administration of endotoxin to rats, in comparison with earlier findings. ALI was induced by the i.p. administration of Esherichia coli LPS 2 (n = 8) or 3 (n = 5) mg/kg, whereas physiological saline was administered to the control animals (n = 5). Eighteen hours after the LPS injections, the animals were euthanized. The lungs and heart were removed in one block for histological study (hematoxylin and eosin [H&E], periodic acid-Schiff [PAS], Mason's trichrome; light microscopy). The lung tissue injury (bronchial wall, vessels, alveoli, interstitium) was graded via a scoring system (0 to 3+). The control animals showed intact lung tissue. Ten of the 13 LPS group had bronchus-associated lymphoid tissue (BALT) hyperplasia. Pathological signs of ALI/ARDS, diffuse alveolar damage (DAD) and emphysema, were observed in 5 and 8 cases, respectively. LPS injection induces primarily BALT hyperplasia and also the less characteristic DAD. This rat model is suitable for the investigation not only of ALI/ARDS but also of BALT hyperplasia occurring as a consequence of chronic pulmonary inflammatory processes.
Assuntos
Lesão Pulmonar Aguda/patologia , Lipopolissacarídeos/farmacologia , Síndrome do Desconforto Respiratório/patologia , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Broncopatias/patologia , Modelos Animais de Doenças , Enfisema/etiologia , Hiperplasia/etiologia , Alvéolos Pulmonares/patologia , Ratos , Síndrome do Desconforto Respiratório/induzido quimicamenteRESUMO
Cortical spreading depolarization (SD) involves activation of NMDA receptors and elicit neurovascular unit dysfunction. NMDA cannot trigger SD in newborns, thus its effect on neurovascular function is not confounded by other aspects of SD. The present study investigated if NMDA affected hypercapnia-induced microvascular and electrophysiological responses in the cerebral cortex of newborn pigs. Anesthetized piglets were fitted with cranial windows over the parietal cortex to study hemodynamic and electrophysiological responses to graded hypercapnia before/after topically applied NMDA assessed with laser-speckle contrast imaging and recording of local field potentials (LFP)/neuronal firing, respectively. NMDA increased cortical blood flow (CoBF), suppressed LFP power in most frequency bands but evoked a 2.5 Hz δ oscillation. The CoBF response to hypercapnia was abolished after NMDA and the hypercapnia-induced biphasic changes in δ and θ LFP power were also altered. MK-801 prevented NMDA-induced increases in CoBF and the attenuation of microvascular reactivity to hypercapnia. The neuronal nitric oxide synthase (nNOS) inhibitor (N-(4 S)-4-amino-5-[aminoethyl]aminopentyl-N'-nitroguanidin) also significantly preserved the CoBF response to hypercapnia after NMDA, although it didn't reduce NMDA-induced increases in CoBF. In conclusion, excess activation of NMDA receptors alone can elicit SD-like neurovascular unit dysfunction involving nNOS activity.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Hipercapnia/fisiopatologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Masculino , Suínos , Vasodilatação/efeitos dos fármacosRESUMO
AIMS: Perinatal asphyxia (PA) often results in hypoxic-ischemic encephalopathy (HIE) in term neonates. Introduction of therapeutic hypothermia improved HIE outcome, but further neuroprotective therapies are still warranted. The present study sought to determine the feasibility of the activation of the cytoprotective PI-3-K/Akt and the MAPK/ERK signaling pathways in the subacute phase of HIE development in a translational newborn pig PA/HIE model. MAIN METHODS: Phosphorylated and total levels of Akt and ERK were determined by Western blotting in brain samples obtained from untreated naive, time control, and PA/HIE animals at 24-48h survival (n=3-3-6,respectively). PA (20min) was induced in anesthetized piglets by ventilation with a hypoxic/hypercapnic (6%O220%CO2) gas mixture. Furthermore, we studied the effect of topically administered specific Akt1/2 and MAPK/ERK kinase inhibitors on Akt and ERK phosphorylation (n=4-4) in the cerebral cortex under normoxic conditions. KEY FINDINGS: PA resulted in significant neuronal injury shown by neuropathology assessment of haematoxylin/eosin stained sections. However, there were no significant differences among the groups in the high phosphorylation levels of both ERK and Akt in the cerebral cortex, hippocampus and subcortical structures. However, the Akt1/2 and MAPK/ERK kinase inhibitors significantly reduced cerebrocortical Akt and ERK phosphorylation within 30min. SIGNIFICANCE: The major finding of the present study is that the PI-3-K/Akt and the MAPK/ERK signaling pathways appear to be constitutively active in the piglet brain, and this activation remains unaltered during HIE development. Thus, neuroprotective strategies aiming to activate these pathways to limit apoptotic neuronal death may offer limited efficacy in this translational model.
Assuntos
Córtex Cerebral/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Proteína Oncogênica v-akt/biossíntese , Animais , Animais Recém-Nascidos , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patologia , Pressão Sanguínea/efeitos dos fármacos , Córtex Cerebral/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Frequência Cardíaca/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Neurônios/patologia , Proteína Oncogênica v-akt/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , SuínosRESUMO
Lung function and bronchoalveolar lavage (BAL) fluid are commonly analyzed to assess the severity of lung disease in sacrificed animals. The input impedance of the respiratory system (Z(rs)) was measured and BAL fluid was collected in intubated, anesthetized, mechanically ventilated rats on three occasions 1 week apart. Measurements were performed in control animals (group C), while lung injury was induced in the other group (group LPS) by i.p. injection of lipopolysaccharide (LPS) before the second measurement. The airway resistance (R(aw)), tissue damping (G) and elastance (H) were determined from the Z(rs) spectra. The total cell counts (TC) from 0.3- to 0.4-ml BAL fluid were also determined. R(aw) exhibited no significant change in either group C (-6.7+/-3.6[S.E.]%) or LPS (-0.9+/-3.7%). Reproducible G and H values were obtained in group C (2.5+/-5.3%, -7.0+/-4.4%), while G and H increased in group LPS (18.4+/-6.5%, 14.9+/-13.8%, p<0.05). The changes in TC followed a similar pattern to those observed in G, with no change in group C (-7.9+/-30%), but with a marked increase in group LPS (580+/-456%, p<0.05). The method devised for repeated BAL measurements in another group of rats without intubation and muscle relaxant resulted in similar results in BAL profile. We conclude that longitudinal follow-up of the airway and tissue mechanics and inflammatory cells in the BAL fluid are feasible in rats. The current method allows an early detection of lung injury, even in a relatively mild form.
Assuntos
Lavagem Broncoalveolar/métodos , Pneumopatias/diagnóstico , Pulmão/fisiopatologia , Testes de Função Respiratória , Mecânica Respiratória , Doença Aguda , Resistência das Vias Respiratórias , Animais , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Estudos de Viabilidade , Lipopolissacarídeos , Complacência Pulmonar , Pneumopatias/induzido quimicamente , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Masculino , Ratos , Ratos Wistar , Respiração ArtificialRESUMO
BACKGROUND: The neurovascular unit encompasses the functional interactions of cerebrovascular and brain parenchymal cells necessary for the metabolic homeostasis of neurons. Previous studies indicated marked but only transient (1-4 h) reactive oxygen species-dependent neurovascular dysfunction in newborn pigs after severe hypoxic/ischemic (H/I) stress contributing to the neuronal injury after birth asphyxia. OBJECTIVES: Our major purpose was to determine if neurovascular dysfunction would also occur later, at 24 h after a milder H/I stress. We also tested if the putative hydroxyl radical scavenger hydrogen (H2) exerted neurovascular protection. METHODS: Anesthetized, ventilated piglets were assigned to three groups of 9 animals: time control, asphyxia/reventilation with air, and asphyxia/reventilation with air +2.1% H2 for 4 h. Asphyxia was induced by suspending ventilation for 8 min. Cerebrovascular reactivity (CR) of pial arterioles was determined using closed cranial window/intravital microscopy 24 h after asphyxia to the endothelium-dependent cerebrovascular stimulus hypercapnia, the neuronal function-dependent stimulus N-methyl-D-aspartate (NMDA), norepinephrine, and sodium nitroprusside. The brains were subjected to histopathology. RESULTS: Hemodynamic parameters, blood gases, and core temperature did not differ significantly among the experimental groups. In the early reventilation period, the recovery of electroencephalographic activity was significantly better in H2-treated animals. Asphyxia/reventilation severely attenuated CR to hypercapnia and NMDA; however, reactivity to norepinephrine and sodium nitroprusside were unaltered. H2 fully or partially preserved CR to hypercapnia or NMDA, respectively. Histopathology revealed modest neuroprotection afforded by H2. CONCLUSIONS: Severe stimulus-selective delayed neurovascular dysfunction develops and persists even after mild H/I stress. H2 alleviates this delayed neurovascular dysfunction that can contribute to its neuroprotective effect.
Assuntos
Asfixia Neonatal/tratamento farmacológico , Sequestradores de Radicais Livres/farmacologia , Hidrogênio/farmacologia , Hipóxia-Isquemia Encefálica/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Pia-Máter/irrigação sanguínea , Pia-Máter/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Arteríolas/fisiopatologia , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patologia , Asfixia Neonatal/fisiopatologia , Gasometria , Ondas Encefálicas/efeitos dos fármacos , Modelos Animais de Doenças , Eletroencefalografia , Agonistas de Aminoácidos Excitatórios/farmacologia , Hemodinâmica/efeitos dos fármacos , Radical Hidroxila/metabolismo , Hipercapnia/fisiopatologia , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Pia-Máter/metabolismo , Pia-Máter/patologia , Recuperação de Função Fisiológica , Respiração Artificial , Suínos , Fatores de Tempo , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
We studied repeatedly the development of bronchial hyperreactivity (BHR) and bronchoalveolar lavage fluid (BALF) in rats undergoing different modes of ovalbumin exposures. Treatment was two intraperitoneal injections of ovalbumin in Groups 1-3, followed by one ovalbumin aerosolization in Groups 2 and 3, while rats in Group 4 received repeated ovalbumin aerosols after one single intraperitoneal injection. BHR was assessed longitudinally on day 0 (before treatment) and on day 14 (Groups 1 and 2) or 20 (Groups 3 and 4) and cellular influx was estimated from BALF. No BHR or change in BALF cellular profile was detected in Groups 1-3. However, the infiltration of inflammatory cells, associated with BHR (PC(100) 8.9+/-1.3 microg/kg vs. 4.2+/-1.1 microg/kg), was observed in Group 4. The BHR was always associated with increased number of eosinophils in the BALF. The substantial interindividual variability confirmed the need for a technique that permits follow-up of lung responsiveness and BALF profile. This approach evidenced strong associations between the severity of BHR and the eosinophilia.
Assuntos
Resistência das Vias Respiratórias/fisiologia , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/fisiopatologia , Líquido da Lavagem Broncoalveolar , Ovalbumina/imunologia , Mecânica Respiratória/fisiologia , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Hiper-Reatividade Brônquica/tratamento farmacológico , Broncoconstritores/farmacologia , Broncoconstritores/uso terapêutico , Contagem de Células/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eosinófilos/efeitos dos fármacos , Cloreto de Metacolina/farmacologia , Cloreto de Metacolina/uso terapêutico , Neutrófilos/efeitos dos fármacos , Ovalbumina/classificação , Ovalbumina/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
AIM: Acetazolamide (AZD) produces cerebral vasodilation. The underlying mechanism is unclear, but it is assumed to be largely due to CO2 retention and acidosis. We tested if cerebrovascular effects of AZD were similar to hypercapnia in the newborn pig. METHODS: We used the closed cranial window/intravital microscopy technique to determine pial arteriolar diameters simultaneously with laser-Doppler flowmetry (LDF) to monitor cortical blood perfusion. Anaesthetized (Na-thiopenthal +alpha-chloralose), ventilated, 1-day-old instrumented piglets (n=38) were divided into five experimental groups: time control (n=11), indomethacin, ibuprofen, Nomega-nitro-L-arginine methyl ester (L-NAME) treatments (1, 30, 15 mg/kg, i.v., n=6, 6, 4, respectively), and global ischaemia/reperfusion (I/R, 10 min induced by elevated intracranial pressure, n=11). Responses to 5-10% inhaled CO2 were recorded before and after the treatments, and then in a similar manner to AZD (10-20 mg/kg, i.v.). RESULTS: Hypercapnia and AZD produced pial arteriolar vasodilation and increases in cortical perfusion. Consistent with previous data, hypercapnia-induced changes were abolished by indomethacin, unaltered by ibuprofen and L-NAME and were significantly attenuated after I/R. AZD-induced vasodilation was also sensitive to indomethacin and I/R and was unaltered by ibuprofen or L-NAME. CONCLUSION: The mechanism of AZD-induced vasodilation appears to be similar/identical to hypercapnia, and pial arteriolar diameter changes reflect changes in cortical perfusion.
Assuntos
Acetazolamida/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Fármacos Cardiovasculares/farmacologia , Indometacina/farmacologia , Isquemia/fisiopatologia , Pia-Máter/irrigação sanguínea , Vasodilatação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Feminino , Fluxometria por Laser-Doppler , Masculino , SuínosRESUMO
Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine implicated in cerebrovascular pathology. The aim of the present study was to characterize the simultaneous effects of an intracarotid administration of TNFalpha on cerebral blood flow (CBF) and the ultrastructure of the blood-brain barrier (BBB) and to determine whether nitric oxide (NO) is a mediator of the TNFalpha-induced alterations in CBF and BBB. TNFalpha (2.5 microg/kg) or saline was infused into the right common carotid artery of male Wistar rats (n = 70). NO production was inhibited with L-NAME (20 mg/kg, i.v.). CBF was monitored for 2 h with laser-Doppler flowmetry. Tissue samples were taken from the unilateral frontoparietal cortex and prepared for electron microscopy. The proportion of capillaries with swollen astrocytic endfeet and the lumen diameter of the capillaries were measured. TNFalpha significantly increased CBF, which reached a maximum of 190% of the baseline 1 h after the cessation of TNFalpha infusion. L-NAME completely prevented the increase in CBF. TNFalpha elevated the swelling of the astrocytic endfeet from a baseline value of 22.4 +/- 9.35% to 64.9 +/- 3.16%. The administration of L-NAME before TNFalpha infusion prevented the astrocytic swelling. These results demonstrate that TNFalpha increases CBF and the swelling of astrocytes through the production of NO. Our data additionally demonstrate that the breakdown of the BBB by circulating TNFalpha may involve the astrocytic endfeet.
Assuntos
Encéfalo/efeitos dos fármacos , Capilares/efeitos dos fármacos , Circulação Cerebrovascular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/ultraestrutura , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/ultraestrutura , Encéfalo/metabolismo , Encéfalo/patologia , Capilares/patologia , Capilares/ultraestrutura , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Inibidores Enzimáticos/farmacologia , Infusões Intra-Arteriais , Fluxometria por Laser-Doppler , Masculino , Microscopia Eletrônica , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
Oscillations in laser Doppler signals derived from the forehead and forearm skin were analyzed in 77 healthy probands from 4 various age groups (ranging between 15 and 77 years) and 22 late-onset sporadic Alzheimer's disease (AD) patients. A characteristic pattern of oscillations in the microcirculatory blood flux ( approximately 8 cycles/min, 0.13 Hz) was observed in the forehead skin, the occurrence of which correlated inversely with age (r = 0.80). The occurrence of forehead vasomotion pattern was 100% in the teenagers, whereas it was significantly less in the elderly control subjects (32%) and in the AD patients (18%). Forearm reactive hyperemia was provoked by 1-min occlusion of the brachial artery, and the vascular reactivity was calculated. This phenomenon also proved to be age-dependent, but the process was not related to AD. Our results indicate that the lack of forehead vasomotion reflects aging better than does the forearm vasomotion. Both of these functions are preserved in AD.