RESUMEN
Perinatal asphyxia (PA) poses a significant threat to multiple organs, particularly the kidneys. Diagnosing PA-associated kidney injury remains challenging, and treatment options are inadequate. Furthermore, there is a lack of long-term follow-up data regarding the renal implications of PA. In this study, 7-day-old male Wistar rats were exposed to PA using a gas mixture (4% O2; 20% CO2 in N2 for 15 min) to investigate molecular pathways linked to renal tubular damage, hypoxia, angiogenesis, heat shock response, inflammation, and fibrosis in the kidney. In a second experiment, adult rats with a history of PA were subjected to moderate renal ischemia-reperfusion (IR) injury to test the hypothesis that PA exacerbates renal susceptibility. Our results revealed an increased gene expression of renal injury markers (kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin), hypoxic and heat shock factors (hypoxia-inducible factor-1α, heat shock factor-1, and heat shock protein-27), proinflammatory cytokines (interleukin-1ß, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1), and fibrotic markers (transforming growth factor-ß, connective tissue growth factor, and fibronectin) promptly after PA. Moreover, a machine learning model was identified through random forest analysis, demonstrating an impressive classification accuracy (95.5%) for PA. Post-PA rats showed exacerbated functional decline and tubular injury and more intense hypoxic, heat shock, proinflammatory, and profibrotic response after renal IR injury compared with controls. In conclusion, PA leads to subclinical kidney injury, which may increase the susceptibility to subsequent renal damage later in life. In addition, the parameters identified through random forest analysis provide a robust foundation for future biomarker research in the context of PA.NEW & NOTEWORTHY This article demonstrates that perinatal asphyxia leads to subclinical kidney injury that permanently increases renal susceptibility to subsequent ischemic injury. We identified major molecular pathways involved in perinatal asphyxia-induced renal complications, highlighting potential targets of therapeutic approaches. In addition, random forest analysis revealed a model that classifies perinatal asphyxia with 95.5% accuracy that may provide a strong foundation for further biomarker research. These findings underscore the importance of multiorgan follow-up for perinatal asphyxia-affected patients.
Asunto(s)
Lesión Renal Aguda , Modelos Animales de Enfermedad , Riñón , Ratas Wistar , Daño por Reperfusión , Animales , Masculino , Lesión Renal Aguda/patología , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/etiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Riñón/patología , Riñón/metabolismo , Fibrosis , Asfixia Neonatal/metabolismo , Asfixia Neonatal/complicaciones , Asfixia Neonatal/patología , Animales Recién Nacidos , Ratas , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Citocinas/metabolismo , Factores de Edad , Mediadores de Inflamación/metabolismoRESUMEN
Neuroinflammation is a salient part of diverse neurological and psychiatric pathologies that associate with neuronal hyperexcitability, but the underlying molecular and cellular mechanisms remain to be identified. Here, we show that peripheral injection of lipopolysaccharide (LPS) renders the dentate gyrus (DG) hyperexcitable to perforant pathway stimulation in vivo and increases the internal spiking propensity of dentate granule cells (DGCs) in vitro 24 h post-injection (hpi). In parallel, LPS leads to a prominent downregulation of chloride extrusion via KCC2 and to the emergence of NKCC1-mediated chloride uptake in DGCs under experimental conditions optimized to detect specific changes in transporter efficacy. These data show that acute neuroinflammation leads to disruption of neuronal chloride regulation, which unequivocally results in a loss of GABAergic inhibition in the DGCs, collapsing the gating function of the DG. The present work provides a mechanistic explanation for neuroinflammation-driven hyperexcitability and consequent cognitive disturbance.
Asunto(s)
Cloruros , Lipopolisacáridos , Humanos , Cloruros/metabolismo , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Enfermedades Neuroinflamatorias , Giro Dentado/metabolismo , Neuronas/metabolismoRESUMEN
SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.
Asunto(s)
Convulsiones , Simportadores de Sodio-Bicarbonato , Niño , Ratones , Humanos , Animales , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Convulsiones/genética , Mutación/genética , Neurotransmisores , Ácido gamma-Aminobutírico/genética , Mamíferos/metabolismo , Antiportadores de Cloruro-Bicarbonato/genética , Antiportadores de Cloruro-Bicarbonato/metabolismoRESUMEN
Systemic inflammation triggers protective as well as pro-inflammatory responses in the brain based on neuronal and/or cytokine signaling, and it associates with acutely and protractedly disrupted cognition. However, the multiple mechanisms underlying the peripheral-central inflammatory signaling are still not fully characterized. We used intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) in freely moving mice with chronically implanted electrodes for recording of local field potentials (LFP) and electrocorticography (ECoG) in the hippocampus and neocortex, respectively. We show here that a sudden switch in the mode of network activity occurred in both areas starting at 10-15 min after the LPS injection, simultaneously with a robust change from exploration to sickness behavior. This switch in cortical mode commenced before any elevations in pro-inflammatory cytokines IL-1ß, TNFα, CCL2 or IL-6 were detected in brain tissue. Thereafter, this mode dominated cortical activity for the recording period of 3 h, except for a partial and transient recovery around 40 min post-LPS. These effects were closely paralleled by changes in ECoG spectral entropy. Continuous recordings for up to 72 h showed a protracted attenuation in hippocampal activity, while neocortical activity recovered after 48 h. The acute sickness behavior recovered by 72 h post-LPS. Notably, urethane (1.3 mg/kg) administered prior to LPS blocked the early effect of LPS on cortical activity. However, experiments under urethane anesthesia which were started 24 h post-LPS (with neuroinflammation fully developed before application of urethane) showed that both theta-supratheta and fast gamma CA1 activity were reduced, DG delta activity was increased, and sharp-wave ripples were abolished. Finally, we observed that experimental compensation of inflammation-induced hypothermia 24-48 h post-LPS promoted seizures and status epilepticus; and that LPS decreased the threshold of kainate-provoked seizures beyond the duration of acute sickness behavior indicating post-acute inflammatory hyperexcitability. Taken together, the strikingly fast development and initial independence of brain cytokines of the LPS-induced cortical mode, its spectral characteristics and simultaneity in hippocampus and neocortex, as well as inhibition by pre-applied urethane, strongly suggest that the underlying mechanisms are based on activation of the afferent vagus nerve and its mainly cholinergic ascending projections to higher brain areas.
Asunto(s)
Citocinas , Conducta de Enfermedad , Ratones , Animales , Citocinas/metabolismo , Lipopolisacáridos/toxicidad , Encéfalo/metabolismo , Inflamación/inducido químicamente , Convulsiones , Uretano/farmacologíaRESUMEN
Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown of Scn2a mRNA in vivo using antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in an Asic1a knock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENT Brain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels. In vivo knockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.
Asunto(s)
Acidosis Respiratoria , Segmento Inicial del Axón , Ratones , Masculino , Animales , Femenino , Dióxido de Carbono , Convulsiones/inducido químicamente , Convulsiones/genética , Células Piramidales , Potenciales de Acción , Ratones Noqueados , ARN MensajeroRESUMEN
The Na-K-2Cl cotransporter NKCC1 is widely expressed in cells within and outside the brain. However, our understanding of its roles in brain functions throughout development, as well as in neuropsychiatric and neurological disorders, has been severely hindered by the lack of reliable data on its developmental and (sub)cellular expression patterns. We provide here the first properly controlled analysis of NKCC1 protein expression in various cell types of the mouse brain using custom-made antibodies and an NKCC1 knock-out validated immunohistochemical procedure, with parallel data based on advanced mRNA approaches. NKCC1 protein and mRNA are expressed at remarkably high levels in oligodendrocytes. In immature neurons, NKCC1 protein was located in the somata, whereas in adult neurons, only NKCC1 mRNA could be clearly detected. NKCC1 immunoreactivity is also seen in microglia, astrocytes, developing pericytes, and in progenitor cells of the dentate gyrus. Finally, a differential expression of NKCC1 splice variants was observed, with NKCC1a predominating in non-neuronal cells and NKCC1b in neurons. Taken together, our data provide a cellular basis for understanding NKCC1 functions in the brain and enable the identification of major limitations and promises in the development of neuron-targeting NKCC1-blockers.
Asunto(s)
Encéfalo , Neuronas , Ratones , Animales , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismo , Hipocampo/metabolismoRESUMEN
In his editorial, Kevin Staley criticizes our recent work demonstrating the lack of effect of bumetanide in a novel model of neonatal seizures. The main points in our response are that (1) our work is on an asphyxia model, not one on "hypercarbia only"; (2) clinically relevant parenteral doses of bumetanide applied in vivo lead to concentrations in the brain parenchyma that are at least an order of magnitude lower than what would be sufficient to exert any direct effect-even a transient one-on neuronal functions, including neonatal seizures; and (3) moreover, bumetanide's molecular target in the brain is the Na-K-2Cl cotransporter NKCC1, which has vital functions in neurons, astrocytes, and oligodendrocytes as well as microglia. This would make it impossible even for highly brain-permeant NKCC1 blockers to specifically target depolarizing and excitatory actions of γ-aminobutyric acid in principal neurons of the brain, which is postulated as the rationale of clinical trials on neonatal seizures.
Asunto(s)
Epilepsia , Enfermedades del Recién Nacido , Bumetanida/uso terapéutico , Epilepsia/tratamiento farmacológico , Humanos , Recién Nacido , Enfermedades del Recién Nacido/tratamiento farmacológico , Convulsiones/tratamiento farmacológico , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1ß (IL-1ß), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.
Asunto(s)
Edema Encefálico/genética , Lesiones Encefálicas/genética , Microglía/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Accidente Cerebrovascular/genética , Animales , Edema Encefálico/inducido químicamente , Edema Encefálico/metabolismo , Edema Encefálico/patología , Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Bumetanida/farmacología , Embrión de Mamíferos , Regulación de la Expresión Génica , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Inflamasomas/efectos de los fármacos , Inflamasomas/metabolismo , Inflamación , Inyecciones Intraventriculares , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolisacáridos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/patología , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Fenotipo , Miembro 2 de la Familia de Transportadores de Soluto 12/deficiencia , Accidente Cerebrovascular/inducido químicamente , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
The Na-K-2Cl cotransporter NKCC1 and the neuron-specific K-Cl cotransporter KCC2 are considered attractive CNS drug targets because altered neuronal chloride regulation and consequent effects on GABAergic signaling have been implicated in numerous CNS disorders. While KCC2 modulators are not yet clinically available, the loop diuretic bumetanide has been used in clinical studies to treat brain disorders and as a tool for NKCC1 inhibition in preclinical models. Bumetanide is known to have anticonvulsant and neuroprotective effects under some pathophysiological conditions. However, as shown in several species from neonates to adults (mice, rats, dogs, and by extrapolation in humans), at the low clinical doses of bumetanide approved for diuresis, this drug has negligible access into the CNS, reaching levels that are much lower than what is needed to inhibit NKCC1 in cells within the brain parenchyma. Several drug discovery strategies have been used over the last â¼15 years to develop brain-permeant compounds that, ideally, should be selective for NKCC1 to eliminate the diuresis mediated by inhibition of renal NKCC2. The strategies employed to improve the pharmacokinetic and pharmacodynamic properties of NKCC1 blockers include evaluation of other clinically approved loop diuretics; development of lipophilic prodrugs of bumetanide; development of side-chain derivatives of bumetanide; and unbiased high-throughput screening approaches of drug discovery based on large chemical compound libraries. The main outcomes are that (1), non-acidic loop diuretics such as azosemide and torasemide may have advantages as NKCC1 inhibitors vs. bumetanide; (2), bumetanide prodrugs achieve significantly higher brain levels of the parent drug and have lower diuretic activity; (3), the novel bumetanide side-chain derivatives do not exhibit any functionally relevant improvement of CNS accessibility or NKCC1 selectivity vs. bumetanide; (4) novel compounds discovered by high-throughput screening may resolve some of the inherent problems of bumetanide, but as yet this has not been achieved. Thus, further research is needed to optimize the design of brain-permeant NKCC1 inhibitors. Another major challenge is to identify the mechanisms whereby various NKCC1-expressing cellular targets of these drug within (e.g., neurons, oligodendrocytes or astrocytes) and outside the brain parenchyma (e.g., blood-brain barrier, choroid plexus, endocrine and immune system), as well as molecular off-target effects, might contribute to their reported therapeutic and adverse effects.
Asunto(s)
Bumetanida/farmacocinética , Sistema Nervioso Central/efectos de los fármacos , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacocinética , Miembro 2 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Animales , HumanosRESUMEN
Apolipoprotein E ε4 allele (APOE4) has been shown to associate with increased susceptibility to SARS-CoV-2 infection and COVID-19 mortality in some previous genetic studies, but information on the role of APOE4 on the underlying pathology and parallel clinical manifestations is scarce. Here we studied the genetic association between APOE and COVID-19 in Finnish biobank, autopsy and prospective clinical cohort datasets. In line with previous work, our data on 2611 cases showed that APOE4 carriership associates with severe COVID-19 in intensive care patients compared with non-infected population controls after matching for age, sex and cardiovascular disease status. Histopathological examination of brain autopsy material of 21 COVID-19 cases provided evidence that perivascular microhaemorrhages are more prevalent in APOE4 carriers. Finally, our analysis of post-COVID fatigue in a prospective clinical cohort of 156 subjects revealed that APOE4 carriership independently associates with higher mental fatigue compared to non-carriers at six months after initial illness. In conclusion, the present data on Finns suggests that APOE4 is a risk factor for severe COVID-19 and post-COVID mental fatigue and provides the first indication that some of this effect could be mediated via increased cerebrovascular damage. Further studies in larger cohorts and animal models are warranted.
Asunto(s)
Apolipoproteína E4/genética , COVID-19/complicaciones , COVID-19/genética , Hemorragia Cerebral/genética , Fatiga Mental/genética , Gravedad del Paciente , Adulto , Anciano , Autopsia , Bancos de Muestras Biológicas , COVID-19/diagnóstico , COVID-19/epidemiología , Hemorragia Cerebral/diagnóstico , Hemorragia Cerebral/epidemiología , Estudios de Cohortes , Femenino , Finlandia/epidemiología , Estudios de Asociación Genética/métodos , Heterocigoto , Humanos , Masculino , Fatiga Mental/diagnóstico , Fatiga Mental/epidemiología , Microvasos/patología , Persona de Mediana Edad , Estudios Prospectivos , Factores de Riesgo , Adulto Joven , Síndrome Post Agudo de COVID-19RESUMEN
OBJECTIVE: Birth asphyxia is a major cause of hypoxic-ischemic encephalopathy (HIE) in neonates and often associated with mortality, neonatal seizures, brain damage, and later life motor, cognitive, and behavioral impairments and epilepsy. Preclinical studies on rodent models are needed to develop more effective therapies for preventing HIE and its consequences. Thus far, the most popular rodent models have used either exposure of intact animals to hypoxia-only, or a combination of hypoxia and carotid occlusion, for the induction of neonatal seizures and adverse outcomes. However, such models lack systemic hypercapnia, which is a fundamental constituent of birth asphyxia with major effects on neuronal excitability. Here, we use a recently developed noninvasive rat model of birth asphyxia with subsequent neonatal seizures to study later life adverse outcome. METHODS: Intermittent asphyxia was induced for 30 min by exposing male and female postnatal day 11 rat pups to three 7 + 3-min cycles of 9% and 5% O2 at constant 20% CO2 . All pups exhibited convulsive seizures after asphyxia. A set of behavioral tests were performed systematically over 14 months following asphyxia, that is, a large part of the rat's life span. Video-electroencephalographic (EEG) monitoring was used to determine whether asphyxia led to the development of epilepsy. Finally, structural brain alterations were examined. RESULTS: The animals showed impaired spatial learning and memory and increased anxiety when tested at an age of 3-14 months. Video-EEG at ~10 months showed an abundance of spontaneous seizures, which was paralleled by neurodegeneration in the hippocampus and thalamus, and by aberrant mossy fiber sprouting. SIGNIFICANCE: The present model of birth asphyxia recapitulates several of the later life consequences associated with human HIE. This model thus allows evaluation of the efficacy of novel therapies designed to prevent HIE and seizures following asphyxia, and of how such therapies might alleviate long-term adverse consequences.
Asunto(s)
Asfixia Neonatal , Disfunción Cognitiva , Epilepsia , Hipoxia-Isquemia Encefálica , Animales , Animales Recién Nacidos , Ansiedad , Asfixia/complicaciones , Asfixia Neonatal/complicaciones , Asfixia Neonatal/tratamiento farmacológico , Encéfalo , Disfunción Cognitiva/complicaciones , Epilepsia/complicaciones , Femenino , Humanos , Hipoxia/complicaciones , Hipoxia-Isquemia Encefálica/complicaciones , Recién Nacido , Masculino , Ratas , Convulsiones/tratamiento farmacológicoRESUMEN
OBJECTIVE: Seizures are common in neonates recovering from birth asphyxia but there is general consensus that current pharmacotherapy is suboptimal and that novel antiseizure drugs are needed. We recently showed in a rat model of birth asphyxia that seizures are triggered by the post-asphyxia recovery of brain pH. Here our aim was to investigate whether carbonic anhydrase inhibitors (CAIs), which induce systemic acidosis, block the post-asphyxia seizures. METHODS: The CAIs acetazolamide (AZA), benzolamide (BZA), and ethoxzolamide (EZA) were administered intraperitoneally or intravenously to 11-day-old rats exposed to intermittent asphyxia (30 min; three 7+3 min cycles of 9% and 5% O2 at 20% CO2 ). Electrode measurements of intracortical pH, Po2 , and local field potentials (LFPs) were made under urethane anesthesia. Convulsive seizures and blood acid-base parameters were examined in freely behaving animals. RESULTS: The three CAIs decreased brain pH by 0.14-0.17 pH units and suppressed electrographic post-asphyxia seizures. AZA, BZA, and EZA differ greatly in their lipid solubility (EZA > AZA > BZA) and pharmacokinetics. However, there were only minor differences in the delay (range 0.8-3.7 min) from intraperitoneal application to their action on brain pH. The CAIs induced a modest post-asphyxia elevation of brain Po2 that had no effect on LFP activity. AZA was tested in freely behaving rats, in which it induced a respiratory acidosis and decreased the incidence of convulsive seizures from 9 of 20 to 2 of 17 animals. SIGNIFICANCE: AZA, BZA, and EZA effectively block post-asphyxia seizures. Despite the differences in their pharmacokinetics, they had similar effects on brain pH, which indicates that their antiseizure mode of action was based on respiratory (hypercapnic) acidosis resulting from inhibition of blood-borne and extracellular vascular carbonic anhydrases. AZA has been used for several indications in neonates, suggesting that it can be safely repurposed for the treatment of neonatal seizures as an add-on to the current treatment regimen.
Asunto(s)
Acidosis , Asfixia Neonatal , Acetazolamida/uso terapéutico , Animales , Asfixia/complicaciones , Asfixia/tratamiento farmacológico , Inhibidores de Anhidrasa Carbónica , Humanos , Recién Nacido , Ratas , Convulsiones/tratamiento farmacológico , Convulsiones/etiologíaRESUMEN
In this response to a commentary by Ben-Ari and Delpire on our recent study on the pharmacology of neonatal seizures in a novel, physiologically validated rat model of birth asphyxia, we wish to rectify their inaccurate descriptions of our model and data. Furthermore, because Ben-Ari and Delpire suggest that negative data on bumetanide from preclinical and clinical trials of neonatal seizures have few implications for (alleged) bumetanide actions on neurons in other brain disorders, we will discuss this topic as well. Based on the poor brain penetration of bumetanide, combined with the extremely wide cellular expression patterns of the target protein NKCC1, it is obvious that the numerous actions of systemically applied bumetanide described in the literature are not mediated by the drug's effects on central neurons.
Asunto(s)
Bumetanida , Epilepsia , Animales , Bumetanida/farmacología , Bumetanida/uso terapéutico , Ratas , Convulsiones/tratamiento farmacológico , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Miembro 2 de la Familia de Transportadores de Soluto 12RESUMEN
Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2 , intracellular carbonic anhydrase (CAi ) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2 -(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.
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Actinas , Anhidrasas Carbónicas , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Anhidrasas Carbónicas/genética , Espinas Dendríticas/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismoRESUMEN
KCC2, best known as the neuron-specific chloride-extruder that sets the strength and polarity of GABAergic currents during neuronal maturation, is a multifunctional molecule that can regulate cytoskeletal dynamics via its C-terminal domain (CTD). We describe the molecular and cellular mechanisms involved in the multiple functions of KCC2 and its splice variants, ranging from developmental apoptosis and the control of early network events to the formation and plasticity of cortical dendritic spines. The versatility of KCC2 actions at the cellular and subcellular levels is also evident in mature neurons during plasticity, disease, and aging. Thus, KCC2 has emerged as one of the most important molecules that shape the overall neuronal phenotype.
Asunto(s)
Simportadores , Cloruros/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
Increased neuronal expression of the Na-K-2Cl cotransporter NKCC1 has been implicated in the generation of seizures and epilepsy. However, conclusions from studies on the NKCC1-specific inhibitor, bumetanide, are equivocal, which is a consequence of the multiple potential cellular targets and poor brain penetration of this drug. Here, we used Nkcc1 knockout (KO) and wildtype (WT) littermate control mice to study the ictogenic and epileptogenic effects of intrahippocampal injection of kainate. Kainate (0.23 µg in 50 nl) induced limbic status epilepticus (SE) in both KO and WT mice with similar incidence, latency to SE onset, and SE duration, but the number of intermittent generalized convulsive seizures during SE was significantly higher in Nkcc1 KO mice, indicating increased SE severity. Following SE, spontaneous recurrent seizures (SRS) were recorded by continuous (24/7) video/EEG monitoring at 0-1, 4-5, and 12-13 weeks after kainate, using depth electrodes in the ipsilateral hippocampus. Latency to onset of electrographic SRS and the incidence of electrographic SRS were similar in WT and KO mice. However, the frequency of electrographic seizures was lower whereas the frequency of electroclinical seizures was higher in Nkcc1 KO mice, indicating a facilitated progression from electrographic to electroclinical seizures during chronic epilepsy, and a more severe epileptic phenotype, in the absence of NKCC1. The present findings suggest that NKCC1 is dispensable for the induction, progression and manifestation of epilepsy, and they do not support the widely held notion that inhibition of NKCC1 in the brain is a useful strategy for preventing or modifying epilepsy.
Asunto(s)
Epilepsia del Lóbulo Temporal/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Femenino , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FenotipoRESUMEN
OBJECTIVE: Neonatal seizures are the most frequent type of neurological emergency in newborn infants, often being a consequence of prolonged perinatal asphyxia. Phenobarbital is currently the most widely used antiseizure drug for treatment of neonatal seizures, but fails to stop them in ~50% of cases. In a neonatal hypoxia-only model based on 11-day-old (P11) rats, the NKCC1 inhibitor bumetanide was reported to potentiate the antiseizure activity of phenobarbital, whereas it was ineffective in a human trial in neonates. The aim of this study was to evaluate the effect of clinically relevant doses of bumetanide as add-on to phenobarbital on neonatal seizures in a noninvasive model of birth asphyxia in P11 rats, designed for better translation to the human term neonate. METHODS: Intermittent asphyxia was induced for 30 minutes by exposing the rat pups to three 7 + 3-minute cycles of 9% and 5% O2 at constant 20% CO2 . Drug treatments were administered intraperitoneally either before or immediately after asphyxia. RESULTS: All untreated rat pups had seizures within 10 minutes after termination of asphyxia. Phenobarbital significantly blocked seizures when applied before asphyxia at 30 mg/kg but not 15 mg/kg. Administration of phenobarbital after asphyxia was ineffective, whereas midazolam (0.3 or 1 mg/kg) exerted significant antiseizure effects when administered before or after asphyxia. In general, focal seizures were more resistant to treatment than generalized convulsive seizures. Bumetanide (0.3 mg/kg) alone or in combination with phenobarbital (15 or 30 mg/kg) exerted no significant effect on seizure occurrence. SIGNIFICANCE: The data demonstrate that bumetanide does not increase the efficacy of phenobarbital in a model of birth asphyxia, which is consistent with the negative data of the recent human trial. The translational data obtained with the novel rat model of birth asphyxia indicate that it is a useful tool to evaluate novel treatments for neonatal seizures.
Asunto(s)
Asfixia Neonatal/tratamiento farmacológico , Bumetanida/uso terapéutico , Modelos Animales de Enfermedad , Midazolam/uso terapéutico , Fenobarbital/uso terapéutico , Convulsiones/tratamiento farmacológico , Animales , Animales Recién Nacidos , Anticonvulsivantes/uso terapéutico , Asfixia Neonatal/complicaciones , Asfixia Neonatal/fisiopatología , Femenino , Hipnóticos y Sedantes/uso terapéutico , Masculino , Ratas , Ratas Wistar , Convulsiones/etiología , Convulsiones/fisiopatología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Resultado del TratamientoRESUMEN
OBJECTIVE: Birth asphyxia (BA) is often associated with seizures that may exacerbate the ensuing hypoxic-ischemic encephalopathy. In rodent models of BA, exposure to hypoxia is used to evoke seizures, that commence already during the insult. This is in stark contrast to clinical BA, in which seizures are typically seen upon recovery. Here, we introduce a term-equivalent rat model of BA, in which seizures are triggered after exposure to asphyxia. METHODS: Postnatal day 11-12 male rat pups were exposed to steady asphyxia (15 min; air containing 5% O2 + 20% CO2 ) or to intermittent asphyxia (30 min; three 5 + 5-min cycles of 9% and 5% O2 at 20% CO2 ). Cortical activity and electrographic seizures were recorded in freely behaving animals. Simultaneous electrode measurements of intracortical pH, Po2 , and local field potentials (LFPs) were made under urethane anesthesia. RESULTS: Both protocols decreased blood pH to <7.0 and brain pH from 7.3 to 6.7 and led to a fall in base excess by 20 mmol·L-1 . Electrographic seizures with convulsions spanning the entire Racine scale were triggered after intermittent but not steady asphyxia. In the presence of 20% CO2 , brain Po2 was only transiently affected by 9% ambient O2 but fell below detection level during the steps to 5% O2 , and LFP activity was nearly abolished. Post-asphyxia seizures were strongly suppressed when brain pH recovery was slowed down by 5% CO2 . SIGNIFICANCE: The rate of brain pH recovery has a strong influence on post-asphyxia seizure propensity. The recurring hypoxic episodes during intermittent asphyxia promote neuronal excitability, which leads to seizures only after the suppressing effect of the hypercapnic acidosis is relieved. The present rodent model of BA is to our best knowledge the first one in which, consistent with clinical BA, behavioral and electrographic seizures are triggered after and not during the BA-mimicking insult.
Asunto(s)
Asfixia/fisiopatología , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Hipoxia/fisiopatología , Animales , Animales Recién Nacidos , Asfixia/etiología , Hipoxia/complicaciones , Masculino , Ratas , Ratas Wistar , Reproducibilidad de los ResultadosRESUMEN
Ionotropic GABA transmission is mediated by anion (mainly Cl-)-permeable GABAA receptors (GABAARs). In immature neurons, GABA exerts depolarizing and sometimes functionally excitatory actions, based on active uptake of Cl- by the Na-K-2Cl cotransporter NKCC1. While functional evidence firmly shows NKCC1-mediated ion transport in immature and diseased neurons, molecular detection of NKCC1 in the brain has turned out to be extremely difficult. In this review, we describe the highly inconsistent data that are available on the cell type-specific expression patterns of the NKCC1 mRNA and protein in the CNS. We discuss the major technical caveats, including a lack of knock-out-controlled immunohistochemistry in the forebrain, possible effects of alternative splicing on the binding of antibodies and RNA probes, and the wide expression of NKCC1 in different cell types, which make whole-tissue analyses of NKCC1 useless for studying its neuronal expression. We also review novel single-cell RNAseq data showing that most of the NKCC1 in the adult CNS may, in fact, be expressed in non-neuronal cells, especially in glia. As future directions, we suggest single-cell NKCC1 mRNA and protein analyses and the use of genetically tagged endogenous proteins or systematically designed novel antibodies, together with proper knock-out controls, for the visualization of endogenous NKCC1 in distinct brain cell types and their subcellular compartments.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Empalme Alternativo , Animales , Sistema Nervioso Central/metabolismo , Cloruros/metabolismo , Epilepsia/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Transporte Iónico , Ratones , Neuroglía/metabolismo , Neuronas/metabolismo , Prosencéfalo , ARN Mensajero/metabolismo , RNA-Seq , Ratas , Receptores de GABA-A/metabolismo , Simportadores/metabolismo , Ácido gamma-AminobutíricoRESUMEN
Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a neurodegenerative disorder caused by loss-of-function mutations in the cystatin B (CSTB) gene. Progression of the clinical symptoms in EPM1 patients, including stimulus-sensitive myoclonus, tonic-clonic seizures, and ataxia, are well described. However, the cellular dysfunction during the presymptomatic phase that precedes the disease onset is not understood. CSTB deficiency leads to alterations in GABAergic signaling, and causes early neuroinflammation followed by progressive neurodegeneration in brains of a mouse model, manifesting as progressive myoclonus and ataxia. Here, we report the first proteome atlas from cerebellar synaptosomes of presymptomatic Cstb-deficient mice, and propose that early mitochondrial dysfunction is important to the pathogenesis of altered synaptic function in EPM1. A decreased sodium- and chloride dependent GABA transporter 1 (GAT-1) abundance was noted in synaptosomes with CSTB deficiency, but no functional difference was seen between the two genotypes in electrophysiological experiments with pharmacological block of GAT-1. Collectively, our findings provide novel insights into the early onset and pathogenesis of CSTB deficiency, and reveal greater complexity to the molecular pathogenesis of EPM1.