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Hypoxic-ischemic encephalopathy (HIE) in neonates causes mortality and neurologic morbidity, including poor cognition with a complex neuropathology. Injury to the cholinergic basal forebrain and its rich innervation of cerebral cortex may also drive cognitive pathology. It is uncertain whether genes associated with adult cognition-related neurodegeneration worsen outcomes after neonatal HIE. We hypothesized that neocortical damage caused by neonatal HI in mice is ushered by persistent cholinergic innervation and interneuron (IN) pathology that correlates with cognitive outcome and is exacerbated by genes linked to Alzheimer's disease. We subjected non-transgenic (nTg) C57Bl6 mice and mice transgenically (Tg) expressing human mutant amyloid precursor protein (APP-Swedish variant) and mutant presenilin (PS1-ΔE9) to the Rice-Vannucci HI model on postnatal day 10 (P10). nTg and Tg mice with sham procedure were controls. Visual discrimination (VD) was tested for cognition. Cortical and hippocampal cholinergic axonal and IN pathology and Aß plaques, identified by immunohistochemistry for choline acetyltransferase (ChAT) and 6E10 antibody respectively, were counted at P210. Simple ChAT+ axonal swellings were present in all sham and HI groups; Tg mice had more than their nTg counterparts, but HI did not affect the number of axonal swellings in APP/PS1 Tg mice. In contrast, complex ChAT+ neuritic clusters (NC) occurred only in Tg mice; HI increased that burden. The abundance of ChAT+ clusters in specific regions correlated with decreased VD. The frequency of attritional ChAT+ INs in the entorhinal cortex (EC) was increased in Tg shams relative to their nTg counterparts, but HI obviated this difference. Cholinergic IN pathology in EC correlated with NC number. The Aß deposition in APP/PS1 Tg mice was not exacerbated by HI, nor did it correlate with other metrics. Adult APP/PS1 Tg mice have significant cortical cholinergic axon and EC ChAT+ IN pathologies; some pathology was exacerbated by neonatal HI and correlated with VD. Mechanisms of neonatal HI induced cognitive deficits and cortical neuropathology may be modulated by genetic risk, perhaps accounting for some of the variability in outcomes.
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Neonatal hypoxic-ischemic encephalopathy (HIE) causes significant morbidity despite treatment with therapeutic hypothermia. Mitochondrial dysfunction may drive the mechanisms underlying neuronal cell death, thereby making mitochondria prime targets for neuroprotection. The mitochondrial permeability transition pore (mPTP) is one such target within mitochondria. In adult animal models, mPTP inhibition is neuroprotective. However, evidence for mPTP inhibition in neonatal models of neurologic disease is less certain. We tested the therapeutic efficacy of the mPTP small molecule inhibitor GNX-4728 and examined the developmental presence of brain mPTP proteins for drug targeting in a neonatal piglet model of hypoxic-ischemic brain injury. Male neonatal piglets were randomized to hypoxia-ischemia (HI) or sham procedure with GNX-4728 (15 mg/kg, IV) or vehicle (saline/cyclodextrin/DMSO, IV). GNX-4728 was administered as a single dose within 5 min after resuscitation from bradycardic arrest. Normal, ischemic, and injured neurons were counted in putamen and somatosensory cortex using hematoxylin and eosin staining. In separate neonatal and juvenile pigs, western blots of putamen mitochondrial-enriched fractions were used to evaluate mitochondrial integrity and the presence of mPTP proteins. We found that a single dose of GNX-4728 did not protect putamen and cortical neurons from cell death after HI. However, loss of mitochondrial matrix integrity occurred within 6h after HI, and while mPTP components are present in the neonatal brain their levels were significantly different compared to that of a mature juvenile brain. Thus, the neonatal brain mPTP may not be a good target for current neurotherapeutic drugs that are developed based on adult mitochondria.
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Asfixia Neonatal/prevención & control , Hipoxia-Isquemia Encefálica/prevención & control , Poro de Transición de la Permeabilidad Mitocondrial , Fármacos Neuroprotectores/uso terapéutico , Animales , Animales Recién Nacidos , Muerte Celular , Paro Cardíaco , Masculino , Putamen/patología , Corteza Somatosensorial/patología , PorcinosRESUMEN
BACKGROUND: Disruption of brain oxygen delivery and consumption after hypoxic-ischemic injury contributes to neonatal mortality and neurological impairment. Measuring cerebral hemodynamic parameters, including cerebral blood flow (CBF), oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO2 ), is clinically important. PURPOSE: Phase-contrast (PC), velocity-selective arterial spin labeling (VSASL), and T2 -relaxation-under-phase-contrast (TRUPC) are magnetic resonance imaging (MRI) techniques that have shown promising results in assessing cerebral hemodynamics in humans. We aimed to test their feasibility in quantifying CBF, OEF, and CMRO2 in piglets. STUDY TYPE: Prospective. ANIMAL MODEL: Ten neonatal piglets subacutely recovered from global hypoxia-ischemia (N = 2), excitotoxic brain injury (N = 6), or sham procedure (N = 2). FIELD STRENGTH/SEQUENCE: VSASL, TRUPC, and PC MRI acquired at 3.0 T. ASSESSMENT: Regional CBF was measured by VSASL. Global CBF was quantified by both PC and VSASL. TRUPC assessed OEF at the superior sagittal sinus (SSS) and internal cerebral veins (ICVs). CMRO2 was calculated from global CBF and SSS-derived OEF. End-tidal carbon dioxide (EtCO2 ) levels of the piglets were also measured. Brain damage was assessed in tissue sections postmortem by counting damaged neurons. STATISTICAL TESTS: Spearman correlations were performed to evaluate associations among CBF (by PC or VSASL), OEF, CMRO2 , EtCO2 , and the pathological neuron counts. Paired t-test was used to compare OEF at SSS with OEF at ICV. RESULTS: Global CBF was 32.1 ± 14.9 mL/100 g/minute and 30.9 ± 8.3 mL/100 g/minute for PC and VSASL, respectively, showing a significant correlation (r = 0.82, P < 0.05). OEF was 54.9 ± 8.8% at SSS and 46.1 ± 5.6% at ICV, showing a significant difference (P < 0.05). Global CMRO2 was 79.1 ± 26.2 µmol/100 g/minute and 77.2 ± 12.2 µmol/100 g/minute using PC and VSASL-derived CBF, respectively. EtCO2 correlated positively with PC-based CBF (r = 0.81, P < 0.05) but negatively with OEF at SSS (r = -0.84, P < 0.05). Relative CBF of subcortical brain regions and OEF at ICV did not significantly correlate, respectively, with the ratios of degenerating-to-total neurons (P = 0.30, P = 0.10). DATA CONCLUSION: Non-contrast MRI can quantify cerebral hemodynamic parameters in normal and brain-injured neonatal piglets. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 2.
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Circulación Cerebrovascular , Consumo de Oxígeno , Animales , Encéfalo/diagnóstico por imagen , Hemodinámica , Humanos , Imagen por Resonancia Magnética , Oxígeno , Estudios Prospectivos , PorcinosRESUMEN
BACKGROUND: Cardiac arrest (CA) is the most common cause of acute neurologic insult in children. Many survivors have significant neurocognitive deficits at 1 year of recovery. Epoxyeicosatrienoic acids (EETs) are multifunctional endogenous lipid signaling molecules that are involved in brain pathobiology and may be therapeutically relevant. However, EETs are rapidly metabolized to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH), limiting their bioavailability. We hypothesized that sEH inhibition would improve outcomes after CA in an infant swine model. Male piglets (3-4 kg, 2 weeks old) underwent hypoxic-asphyxic CA. After resuscitation, they were randomized to intravenous treatment with an sEH inhibitor (TPPU, 1 mg/kg; n = 8) or vehicle (10% poly(ethylene glycol); n = 9) administered at 30 min and 24 h after return of spontaneous circulation. Two sham-operated groups received either TPPU (n = 9) or vehicle (n = 8). Neurons were counted in hematoxylin- and eosin-stained sections from putamen and motor cortex in 4-day survivors. RESULTS: Piglets in the CA + vehicle groups had fewer neurons than sham animals in both putamen and motor cortex. However, the number of neurons after CA did not differ between vehicle- and TPPU-treated groups in either anatomic area. Further, 20% of putamen neurons in the Sham + TPPU group had abnormal morphology, with cell body attrition and nuclear condensation. TPPU treatment also did not reduce neurologic deficits. CONCLUSION: Treatment with an sEH inhibitor at 30 min and 24 h after resuscitation from asphyxic CA does not protect neurons or improve acute neurologic outcomes in piglets.
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Inhibidores Enzimáticos/uso terapéutico , Epóxido Hidrolasas/antagonistas & inhibidores , Paro Cardíaco/complicaciones , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/etiología , Animales , Asfixia/patología , Muerte Celular , Estrés del Retículo Endoplásmico , Masculino , Corteza Motora/patología , Neuronas/patología , Compuestos de Fenilurea/uso terapéutico , Piperidinas/uso terapéutico , Putamen/patología , Porcinos , Resultado del TratamientoRESUMEN
BACKGROUND: Diffusion MRI is routinely used to evaluate brain injury in neonatal encephalopathy. Although abnormal mean diffusivity (MD) is often attributed to cytotoxic edema, the specific contribution from neuronal pathology is unclear. PURPOSE: To determine whether MD from high-resolution diffusion tensor imaging (DTI) can detect variable degrees of neuronal degeneration and pathology in piglets with brain injury induced by excitotoxicity or global hypoxia-ischemia (HI) with or without overt infarction. STUDY TYPE: Prospective. ANIMAL MODEL: Excitotoxic brain injury was induced in six neonatal piglets by intrastriatal stereotaxic injection of the glutamate receptor agonist quinolinic acid (QA). Three piglets underwent global HI or a sham procedure. Piglets recovered for 20-96 hours before undergoing MRI (n = 9). FIELD STRENGTH/SEQUENCE: 3.0T MRI with DTI, T1 - and T2 -weighted imaging. ASSESSMENT: MD, fractional anisotropy (FA), and qualitative T2 injury were assessed in the putamen and caudate. The cell bodies of normal neurons, degenerating neurons (excitotoxic necrosis, ischemic necrosis, or necrosis-apoptosis cell death continuum), and injured neurons with equivocal degeneration were counted by histopathology. STATISTICAL TESTS: Spearman correlations were used to compare MD and FA to normal, degenerating, and injured neurons. T2 injury and neuron counts were evaluated by descriptive analysis. RESULTS: The QA insult generated titratable levels of neuronal pathology. In QA, HI, and sham piglets, lower MD correlated with higher ratios of degenerating-to-total neurons (P < 0.05), lower ratios of normal-to-total neurons (P < 0.05), and greater numbers of degenerating neurons (P < 0.05). MD did not correlate with abnormal neurons exhibiting nascent injury (P > 0.99). Neuron counts were not related to FA (P > 0.30) or to qualitative injury from T2 -weighted MRI. DATA CONCLUSION: MD is more accurate than FA for detecting neuronal degeneration and loss during acute recovery from neonatal excitotoxic and HI brain injury. MD does not reliably detect nonfulminant, nascent, and potentially reversible neuronal injury. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2 J. Magn. Reson. Imaging 2020;52:1216-1226.
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Imagen de Difusión por Resonancia Magnética , Imagen de Difusión Tensora , Animales , Muerte Celular , Neuronas , Proyectos Piloto , Estudios Prospectivos , PorcinosRESUMEN
Therapeutic hypothermia is the standard of clinical care for moderate neonatal hypoxic-ischemic encephalopathy. We investigated the independent and interactive effects of hypoxia-ischemia (HI) and temperature on neuronal survival and injury in basal ganglia and cerebral cortex in neonatal piglets. Male piglets were randomized to receive HI injury or sham procedure followed by 29 h of normothermia, sustained hypothermia induced at 2 h, or hypothermia with rewarming during fentanyl-nitrous oxide anesthesia. Viable and injured neurons and apoptotic profiles were counted in the anterior putamen, posterior putamen, and motor cortex at 29 h after HI injury or sham procedure. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) identified genomic DNA fragmentation to confirm cell death. Though hypothermia after HI preserved viable neurons in the anterior and posterior putamen, hypothermia prevented neuronal injury in only the anterior putamen. Hypothermia initiated 2 h after injury did not protect against apoptotic cell death in either the putamen or motor cortex, and rewarming from hypothermia was associated with increased apoptosis in the motor cortex. In non-HI shams, sustained hypothermia during anesthesia was associated with neuronal injury and corresponding viable neuron loss in the anterior putamen and motor cortex. TUNEL confirmed increased neurodegeneration in the putamen of hypothermic shams. Anesthetized, normothermic shams did not show abnormal neuronal cytopathology in the putamen or motor cortex, thereby demonstrating minimal contribution of the anesthetic regimen to neuronal injury during normothermia. We conclude that the efficacy of hypothermic protection after HI is region specific and that hypothermia during anesthesia in the absence of HI may be associated with neuronal injury in the developing brain. Studies examining the potential interactions between hypothermia and anesthesia, as well as with longer durations of hypothermia, are needed.
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Hipotermia Inducida , Hipoxia-Isquemia Encefálica/patología , Neuronas/patología , Animales , Animales Recién Nacidos , Hipotermia Inducida/efectos adversos , Hipotermia Inducida/métodos , Masculino , PorcinosRESUMEN
Delayed hippocampal injury and memory impairments follow neonatal hypoxia-ischemia (HI) despite the use of therapeutic hypothermia (TH). Death of hippocampal pyramidal cells occurs acutely after HI, but characterization of delayed cell death and injury of interneurons (INs) is unknown. We hypothesize that injury of INs after HI is: (i) asynchronous to that of pyramidal cells, (ii) independent of injury severity, and (iii) unresponsive to TH. HI was induced in C57BL6 mice at p10 with unilateral right carotid ligation and 45 min of hypoxia (FiO2 = 0.08). Mice were randomized to normothermia (36 °C, NT) or TH (31 °C) for 4 hr after HI and anesthesia-exposed shams were use as controls. Brains were studied at 24 hr (p11) or 8 days (p18) after HI. Vglut1, GAD65/67, PSD95, parvalbumin (PV) and calbindin-1 (Calb1) were measured. Cell death was assessed using cresyl violet staining and TUNEL assay. Hippocampal atrophy and astroglyosis at p18 were used to assess injury severity and to correlate with number of PV + INs. VGlut1 level decreased by 30% at 24 hr after HI, while GAD65/67 level decreased by â¼50% in forebrain 8 days after HI, a decrease localized in CA1 and CA3. PSD95 levels decreased in forebrain by 65% at 24 hr after HI and remained low 8 days after HI. PV + INs increased in numbers (per mm2 ) and branching between p11 and p18 in sham mice but not in NT and TH mice, resulting in 21-52% fewer PV + INs in injured mice at p18. Calb1 protein and mRNA were also reduced in HI injured mice at p18. At p18, somatodendritic attrition of INs was evident in all injured mice without evidence of cell death. Neither hippocampal atrophy nor astroglyosis correlated with the number of PV + INs at p18. Thus, HI exposure has long lasting effects in the hippocampus impairing the development of the GABAergic system with only partial protection by TH independent of the degree of hippocampal injury. © 2018 Wiley Periodicals, Inc.
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Hipocampo/patología , Hipotermia Inducida/métodos , Hipoxia-Isquemia Encefálica/terapia , Interneuronas/patología , Animales , Animales Recién Nacidos , Calbindina 1/genética , Calbindina 1/metabolismo , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large/metabolismo , Lateralidad Funcional , Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/metabolismo , Hipoxia-Isquemia Encefálica/patología , Ratones , Ratones Endogámicos C57BL , Parvalbúminas/metabolismo , Tubulina (Proteína)/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motoneurons. Hyperexcitability and excitotoxicity have been implicated in the early pathogenesis of ALS. Studies addressing excitotoxic motoneuron death and intracellular Ca(2+) overload have mostly focused on Ca(2+) influx through AMPA glutamate receptors. However, intrinsic excitability of motoneurons through voltage-gated ion channels may also have a role in the neurodegeneration. In this study we examined the function and localization of voltage-gated Ca(2+) channels in cultured spinal cord motoneurons from mice expressing a mutant form of human superoxide dismutase-1 with a Gly93âAla substitution (G93A-SOD1). Using whole-cell patch-clamp recordings, we showed that high voltage activated (HVA) Ca(2+) currents are increased in G93A-SOD1 motoneurons, but low voltage activated Ca(2+) currents are not affected. G93A-SOD1 motoneurons also have altered persistent Ca(2+) current mediated by L-type Ca(2+) channels. Quantitative single-cell RT-PCR revealed higher levels of Ca1a, Ca1b, Ca1c, and Ca1e subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the increase of HVA Ca(2+) currents may result from upregulation of Ca(2+) channel mRNA expression in motoneurons. The localizations of the Ca1B N-type and Ca1D L-type Ca(2+) channels in motoneurons were examined by immunocytochemistry and confocal microscopy. G93A-SOD1 motoneurons had increased Ca1B channels on the plasma membrane of soma and dendrites. Ca1D channels are similar on the plasma membrane of soma and lower on the plasma membrane of dendrites of G93A-SOD1 motoneurons. Our study demonstrates that voltage-gated Ca(2+) channels have aberrant functions and localizations in ALS mouse motoneurons. The increased HVA Ca(2+) currents and PCCa current could contribute to early pathogenesis of ALS.
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Esclerosis Amiotrófica Lateral/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Ácido Glutámico/metabolismo , Ratones Transgénicos , Neuronas Motoras/patología , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Therapeutic hypothermia provides incomplete neuroprotection after hypoxia-ischemia (HI)-induced brain injury in neonates. We previously showed that cortical neuron and white matter apoptosis are promoted by hypothermia and early rewarming in a piglet model of HI. The unfolded protein response (UPR) may be one of the potential mediators of this cell death. Here, neonatal piglets underwent HI or sham surgery followed by 29 h of normothermia, 2 h of normothermia + 27 h of hypothermia or 18 h of hypothermia + rewarming. Piglets recovered for 29 h. Immunohistochemistry for endoplasmic reticulum to nucleus signaling-1 protein (ERN1), a marker of UPR activation, was used to determine the ratios of ERN1+ macroglia and neurons in the motor subcortical white matter and cerebral cortex. The ERN1+ macroglia were immunophenotyped as oligodendrocytes and astrocytes by immunofluorescent colabeling. Temperature (p = 0.046) and HI (p < 0.001) independently affected the ratio of ERN1+ macroglia. In sham piglets, sustained hypothermia (p = 0.011) and rewarming (p = 0.004) increased the ERN1+ macroglia ratio above that in normothermia. HI prior to hypothermia diminished the UPR. Ratios of ERN1+ macroglia correlated with white matter apoptotic profile counts in shams (r = 0.472; p = 0.026), thereby associating UPR activation with white matter apoptosis during hypothermia and rewarming. Accordingly, macroglial cell counts decreased in shams that received sustained hypothermia (p = 0.009) or rewarming (p = 0.007) compared to those in normothermic shams. HI prior to hypothermia neutralized the macroglial cell loss. Neither HI nor temperature affected ERN1+ neuron ratios. In summary, delayed hypothermia and rewarming activate the macroglial UPR, which is associated with white matter apoptosis. HI may decrease the macroglial endoplasmic reticulum stress response after hypothermia and rewarming.
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Apoptosis/fisiología , Hipotermia/metabolismo , Hipoxia-Isquemia Encefálica/metabolismo , Neuroglía/metabolismo , Recalentamiento , Respuesta de Proteína Desplegada , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Masculino , Oligodendroglía/metabolismo , Porcinos , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
TDP-43 proteinopathy, initially associated with ALS and FTD, is also found in 30-60% of Alzheimer's disease (AD) cases and correlates with worsened cognition and neurodegeneration. A major component of this proteinopathy is depletion of this RNA-binding protein from the nucleus, which compromises repression of non-conserved cryptic exons in neurodegenerative diseases. To test whether nuclear depletion of TDP-43 may contribute to the pathogenesis of AD cases with TDP-43 proteinopathy, we examined the impact of depletion of TDP-43 in populations of neurons vulnerable in AD, and on neurodegeneration in an AD-linked context. Here, we show that some populations of pyramidal neurons that are selectively vulnerable in AD are also vulnerable to TDP-43 depletion in mice, while other forebrain neurons appear spared. Moreover, TDP-43 depletion in forebrain neurons of an AD mouse model exacerbates neurodegeneration, and correlates with increased prefibrillar oligomeric Aß and decreased Aß plaque burden. These findings support a role for nuclear depletion of TDP-43 in the pathogenesis of AD and provide strong rationale for developing novel therapeutics to alleviate the depletion of TDP-43 and functional antemortem biomarkers associated with its nuclear loss.
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Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Proteínas de Unión al ADN/deficiencia , Enfermedades Neurodegenerativas/etiología , Placa Amiloide , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antineoplásicos Hormonales/uso terapéutico , Autofagia/genética , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/metabolismo , Mitocondrias/patología , Placa Amiloide/etiología , Placa Amiloide/genética , Placa Amiloide/patología , Presenilina-1/genética , Presenilina-1/metabolismo , Prosencéfalo/patología , Tamoxifeno/uso terapéuticoRESUMEN
The severity of perinatal hypoxia-ischemia and the delay in initiating therapeutic hypothermia limit the efficacy of hypothermia. After hypoxia-ischemia in neonatal piglets, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been found to contribute to oxidative stress at 3 h of reoxygenation and to eventual neurodegeneration. We tested whether early administration of a 20-HETE synthesis inhibitor after reoxygenation augments neuroprotection with 3-hour delayed hypothermia. In two hypothermic groups, whole body cooling from 38.5 to 34°C was initiated 3 h after hypoxia-ischemia. Rewarming occurred from 20 to 24 h; then anesthesia was discontinued. One hypothermic group received a 20-HETE inhibitor at 5 min after reoxygenation. A sham-operated group and another hypoxia-ischemia group remained normothermic. At 10 days of recovery, resuscitated piglets with delayed hypothermia alone had significantly greater viable neuronal density in the putamen, caudate nucleus, sensorimotor cortex, CA3 hippocampus, and thalamus than did piglets with normothermic recovery, but the values remained less than those in the sham-operated group. In piglets administered the 20-HETE inhibitor before hypothermia, the density of viable neurons in the putamen, cortex and thalamus was significantly greater than in the group with hypothermia alone. Cytochrome P450 4A, which can synthesize 20-HETE, was expressed in piglet neurons in these regions. We conclude that early treatment with a 20-HETE inhibitor enhances the therapeutic benefit of delayed hypothermia in protecting neurons in brain regions known to be particularly vulnerable to hypoxia-ischemia in term newborns.
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Amidinas/farmacología , Citocromo P-450 CYP4A/metabolismo , Ácidos Hidroxieicosatetraenoicos/antagonistas & inhibidores , Hipotermia Inducida/métodos , Hipoxia-Isquemia Encefálica/terapia , Fármacos Neuroprotectores/farmacología , Amidinas/administración & dosificación , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , PorcinosRESUMEN
PURPOSE: To investigate if frequency-dependent contrasts using oscillating gradient diffusion MRI (dMRI) can detect hypoxia-ischemia (HI) -induced neurodegeneration in the neonatal mouse hippocampus. METHODS: Pulsed- and oscillating-gradient dMR images (at 50, 100, and 150 Hz) were acquired from postmortem fixed brains of mice exposed to neonatal HI using the Rice-Vanucci model. MRI data were acquired at 1, 4, and 8 days following HI, and compared with histological data from the same mice for in situ histological validation of the MRI findings. RESULTS: The rate of change of apparent diffusion coefficient with gradient frequency (Δf ADC) revealed unique layer-specific contrasts in the neonatal mouse hippocampus. Δf ADC measurements were found to show a significant decrease in response to neonatal HI injury, in the pyramidal (Py) and granule (GrDG) cell layers compared with contralateral regions. The areas of reduced intensity in the Δf ADC maps corresponded to regional neurodegeneration seen with H&E and Fluoro-Jade C stainings, indicating that alterations in Δf ADC contrasts are sensitive to early microstructural changes due to HI-induced neurodegeneration in the studied regions. CONCLUSION: The findings show that the frequency-dependence of ADC measurements with oscillating-gradient dMRI can provide a sensitive contrast to detect HI-induced neurodegeneration in neuronal layers of the neonatal mouse hippocampus.
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Imagen de Difusión por Resonancia Magnética/métodos , Hipocampo/patología , Hipoxia-Isquemia Encefálica/patología , Animales , Animales Recién Nacidos , Anisotropía , Técnicas In Vitro , RatonesRESUMEN
PURPOSE: We investigated whether oscillating gradient diffusion MRI (dMRI) can provide information on brain microstructural changes after formaldehyde fixation and after hypoxic-ischemic (HI) injury beyond that provided by conventional dMRI. METHODS: Pulsed gradient spin echo (PGSE) and oscillating gradient spin echo (OGSE) dMRI of the adult mouse brain was performed in vivo (50-200 Hz, b = 600 mm(2)/s), and a similar protocol was applied to neonatal mouse brains at 24 h after unilateral hypoxia-ischemia. Animals were perfusion fixed with 4% paraformaldehyde for ex vivo dMRI and histology. RESULTS: Apparent diffusion coefficients (ADCs) measured in the live adult mouse brain presented tissue-dependent frequency-dependence. In vivo OGSE-ADC maps at high oscillating frequencies (>100 Hz) showed clear contrast between the molecular layer and granule cell layer in the adult mouse cerebellum. Formaldehyde fixation significantly altered the temporal diffusion spectra in several brain regions. In neonatal mouse brains with HI injury, in vivo ADC measurements from edema regions showed diminished edema contrasts at 200 Hz compared with the PGSE results. Histology showed severe tissue swelling and necrosis in the edema regions. CONCLUSION: The results demonstrate the unique ability of OGSE-dMRI in delineating tissue microstructures at different spatial scales.
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Cerebelo/patología , Imagen de Difusión por Resonancia Magnética/métodos , Hipoxia-Isquemia Encefálica/patología , Oscilometría/métodos , Animales , Animales Recién Nacidos , Imagen Eco-Planar , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Ratones Endogámicos C57BLRESUMEN
Introduction: Loss of proteasome function, proteinopathy, and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology. Methods: Using pigs, a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex, we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes. Results: By Western blotting, proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus, PSMB5 immunoreactivity was diffuse, particulate, and clustered, including perinucleolar decorations. By fluorogenic assay, proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex, proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15-20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay, 26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay, MG132-inhibitable proteasome CTL activity was localized to the neuropil, as a mosaic, and to cell bodies, nuclei, and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h, brain proteasome CTL activity was modestly increased. Discussion: This study shows that the proteasome in the pig brain has relative regional uniformity, prominent nuclear and perinuclear presence with catalytic activity, a mitochondrial association with activity, 26S-single cap dominance, and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable.
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Noninvasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent advances in diffusion magnetic resonance imaging enabled in vivo examination of tissue microstructures well beyond the imaging resolution. Here, we proposed to use diffusion time-dependent diffusion kurtosis imaging (tDKI) to simultaneously assess cellular morphology and transmembrane permeability in hypoxic-ischemic (HI) brain injury. Through numerical simulations and organoid imaging, we demonstrated the feasibility of capturing effective size and permeability changes using tDKI. In vivo MRI of HI-injured mouse brains detected a shift of the tDKI peak to longer diffusion times, suggesting swelling of the cellular processes. Furthermore, we observed a faster decrease of the tDKI tail, reflecting increased transmembrane permeability associated with up-regulated water exchange or necrosis. Such information, unavailable from a single diffusion time, can predict salvageable tissues. Preliminary applications of tDKI in patients with ischemic stroke suggested increased transmembrane permeability in stroke regions, illustrating tDKI's potential for detecting pathological changes in the clinics.
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Isquemia Encefálica , Imagen de Difusión por Resonancia Magnética , Animales , Imagen de Difusión por Resonancia Magnética/métodos , Ratones , Humanos , Isquemia Encefálica/diagnóstico por imagen , Isquemia Encefálica/patología , Isquemia Encefálica/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Encéfalo/metabolismo , Modelos Animales de Enfermedad , MasculinoRESUMEN
Polynitroxylated PEGylated hemoglobin (PNPH, aka SanFlow) possesses superoxide dismutase/catalase mimetic activities that may directly protect the brain from oxidative stress. Stabilization of PNPH with bound carbon monoxide prevents methemoglobin formation during storage and permits it to serve as an anti-inflammatory carbon monoxide donor. We determined whether small volume transfusion of hyperoncotic PNPH is neuroprotective in a porcine model of traumatic brain injury (TBI) with and without accompanying hemorrhagic shock (HS). TBI was produced by controlled cortical impact over the frontal lobe of anesthetized juvenile pigs. Hemorrhagic shock was induced starting 5â min after TBI by 30â ml/kg blood withdrawal. At 120â min after TBI, pigs were resuscitated with 60â ml/kg lactated Ringer's (LR) or 10 or 20â ml/kg PNPH. Mean arterial pressure recovered to approximately 100 mmHg in all groups. A significant amount of PNPH was retained in the plasma over the first day of recovery. At 4 days of recovery in the LR-resuscitated group, the volume of frontal lobe subcortical white matter ipsilateral to the injury was 26.2 ± 7.6% smaller than homotypic contralateral volume, whereas this white matter loss was only 8.6 ± 12.0% with 20-ml/kg PNPH resuscitation. Amyloid precursor protein punctate accumulation, a marker of axonopathy, increased in ipsilateral subcortical white matter by 132 ± 71% after LR resuscitation, whereas the changes after 10â ml/kg (36 ± 41%) and 20â ml/kg (26 ± 15%) PNPH resuscitation were not significantly different from controls. The number of cortical neuron long dendrites enriched in microtubules (length >50 microns) decreased in neocortex by 41 ± 24% after LR resuscitation but was not significantly changed after PNPH resuscitation. The perilesion microglia density increased by 45 ± 24% after LR resuscitation but was unchanged after 20â ml/kg PNPH resuscitation (4 ± 18%). Furthermore, the number with an activated morphology was attenuated by 30 ± 10%. In TBI pigs without HS followed 2â h later by infusion of 10â ml/kg LR or PNPH, PNPH remained neuroprotective. These results in a gyrencephalic brain show that resuscitation from TBI + HS with PNPH protects neocortical gray matter, including dendritic microstructure, and white matter axons and myelin. This neuroprotective effect persists with TBI alone, indicating brain-targeting benefits independent of blood pressure restoration.
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Non-invasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent developments in diffusion MRI have produced new tools for examining tissue microstructure at a level well below the imaging resolution. Here, we report the use of diffusion time ( t )-dependent diffusion kurtosis imaging ( t DKI) to simultaneously assess the morphology and transmembrane permeability of cells and their processes in the context of pathological changes in hypoxic-ischemic brain (HI) injury. Through Monte Carlo simulations and cell culture organoid imaging, we demonstrate feasibility in measuring effective size and permeability changes based on the peak and tail of t DKI curves. In a mouse model of HI, in vivo imaging at 11.7T detects a marked shift of the t DKI peak to longer t in brain edema, suggesting swelling and beading associated with the astrocytic processes and neuronal neurites. Furthermore, we observed a faster decrease of the t DKI tail in injured brain regions, reflecting increased membrane permeability that was associated with upregulated water exchange upon astrocyte activation at acute stage as well as necrosis with disrupted membrane integrity at subacute stage. Such information, unavailable with conventional diffusion MRI at a single t, can predict salvageable tissues. For a proof-of-concept, t DKI at 3T on an ischemic stroke patient suggested increased membrane permeability in the stroke region. This work therefore demonstrates the potential of t DKI for in vivo detection of the pathological changes in microstructural morphology and transmembrane permeability after ischemic injury using a clinically translatable protocol.
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The effects of hypothermia on neonatal encephalopathy may vary topographically and cytopathologically in the neocortex with manifestations potentially influenced by seizures that alter the severity, distribution, and type of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia (HT) with continuous electroencephalography (cEEG) for seizures. Neonatal male piglets received HI-normothermia (NT), HI-HT, sham-NT, or sham-HT treatments. Randomized unmedicated sham and HI piglets underwent cEEG during recovery. Survival was 2-7 days. Normal and pathological neurons were counted in different neocortical areas, identified by cytoarchitecture and connectomics, using hematoxylin and eosin staining and immunohistochemistry for RNA-binding FOX-1 homolog 3 (Rbfox3/NeuN). Seizure burden was determined. HI-NT piglets had a reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to sham-NT and sham-HT piglets with differing severities in the anterior and posterior motor, somatosensory, and frontal cortices. Neocortical neuropathology was attenuated by HT. HT protection was prominent in layer III of the inferior parietal cortex. Rbfox3 immunoreactivity distinguished cortical neurons as: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had an increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT piglets. Neuronal Rbfox3 depletion was partly rescued by HT. Seizure burdens in HI-NT and HI-HT piglets were similar. We conclude that the neonatal HI piglet neocortex has: (1) suprasylvian vulnerability to HI and seizures; (2) a limited neuronal cytopathological repertoire in functionally different regions that engages protective mechanisms with HT; (3) higher seizure burden, insensitive to HT, that is correlated with more panlaminar ischemic-necrotic neurons in the somatosensory cortex; and (4) pathological RNA splicing protein nuclear depletion that is sensitive to HT. This work demonstrates that HT protection of the neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores neuronal depletion of RNA splicing factor.
Asunto(s)
Hipotermia , Hipoxia-Isquemia Encefálica , Neocórtex , Animales , Masculino , Porcinos , Hipotermia/patología , Animales Recién Nacidos , Neocórtex/metabolismo , Hipoxia/patología , Neuronas/metabolismo , Isquemia/patología , Hipoxia-Isquemia Encefálica/patología , ConvulsionesRESUMEN
Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93âAla substitution (G93A-SOD1). In this study, we have examined, using whole-cell patch-clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing enhanced green fluorescent protein (eGFP) driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP-expressing (Hb9-eGFP(+)) neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP(+) motoneurons prepared from G93A-SOD1 embryos and from their nontransgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature IPSCs (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell reverse transcription-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Modelos Animales de Enfermedad , Neuronas Motoras/metabolismo , Receptores de Glicina/genética , Médula Espinal/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Células Cultivadas , Regulación hacia Abajo/genética , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/enzimología , Neuronas Motoras/patología , Inhibición Neural/genética , Receptores de Glicina/antagonistas & inhibidores , Receptores de Glicina/metabolismo , Médula Espinal/enzimología , Médula Espinal/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
Ablation of mouse occipital cortex induces precisely timed and uniform p53-modulated and Bax-dependent apoptosis of thalamocortical projection neurons in the dorsal lateral geniculate nucleus (LGN) by 7 d after lesion. We tested the hypothesis that this neuronal apoptosis is initiated by oxidative stress and the mitochondrial permeability transition pore (mPTP). Preapoptotic LGN neurons accumulate mitochondria, Zn(2+) and Ca(2+), and generate higher levels of reactive oxygen species (ROS), including superoxide, nitric oxide (NO), and peroxynitrite, than LGN neurons with an intact cortical target. Preapoptosis of LGN neurons is associated with increased formation of protein carbonyls, protein nitration, and protein S-nitrosylation. Genetic deletion of nitric oxide synthase 1 (nos1) and inhibition of NOS1 with nitroindazole protected LGN neurons from apoptosis, revealing NO as a mediator. Putative components of the mPTP are expressed in mouse LGN, including the voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (CyPD). Nitration of CyPD and ANT in LGN mitochondria occurs by 2 d after cortical injury. Chemical cross-linking showed that LGN neuron preapoptosis is associated with formation of CyPD and VDAC oligomers, consistent with mPTP formation. Mice without CyPD are rescued from neuron apoptosis as are mice treated with the mPTP inhibitors TRO-19622 (cholest-4-en-3-one oxime) and TAT-Bcl-X(L)-BH4. Manipulation of the mPTP markedly attenuated the early preapoptotic production of reactive oxygen/nitrogen species in target-deprived neurons. Our results demonstrate in adult mouse brain neurons that the mPTP functions to enhance ROS production and the mPTP and NO trigger apoptosis; thus, the mPTP is a target for neuroprotection in vivo.