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Cited by 312Organ Transplantation Techniques and OutcomesTraumatic Brain Injury and Neurovascular DisturbancesOrgan Donation and Transplantation

The DOI matches its Crossref record; 51 of the 51 references it cites were found in a registry or an index; none of the 15 sources its claims cite is checked for support yet.

Restoration of brain circulation and cellular functions hours post-mortem

Open access
Vrselja, Zvonimir, Daniele, Stefano G., Silbereis, John, Talpo, Francesca, Morozov, Yury M., Sousa, André M. M., Tanaka, Brian S., Skarica, Mario, Pletikos, Mihovil, Kaur, Navjot, Zhuang, Zhen W., Liu, Zhao, Alkawadri, Rafeed, Sinusas, Albert J., Latham, Stephen R., Waxman, Stephen G., Sestan, Nenad·2019·Nature
DOI 10.1038/s41586-019-1099-1PMC PMC6844189
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Abstract

The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports that an extracorporeal pulsatile-perfusion system (BrainEx) with a hemoglobin-based, acellular, cytoprotective perfusate restored microcirculation and selected molecular and cellular functions in intact porcine brains after 4 hours postmortem, maintained for 6 hours of ex vivo normothermic perfusion. It found preserved cytoarchitecture, reduced cell death, restored vascular/glial/synaptic and metabolic activity, but no global electrocorticographic activity.

Key findings

  • After 4 h postmortem and 6 h perfusion (10 h total), BEx-perfused brains showed patent microvasculature, preserved hippocampal/neocortical/cerebellar cytoarchitecture, reduced activated caspase 3, and maintained neuronal, glial, and myelin architecture compared with 10 h PMI and control-perfusate brains.
  • BEx restored vascular dilatory response to nimodipine, glial inflammatory response to LPS (increased IL-1α, IL-1β, IL-6, and IL-8 relative to other conditions), spontaneous excitatory postsynaptic currents, and active cerebral metabolism.
  • At normothermia, average oxygen and glucose expenditure was approximately 2 mL/min/100 g and 6.5 mg/min/100 g tissue, respectively, with an arteriovenous lactate gradient of approximately −1 mmol/L.
  • Hippocampal CA3 pyramidal neurons after BEx perfusion had membrane capacitance 125±43 pF, input resistance 195±63 MΩ, membrane time constant 32.3±8.4 ms, resting potential −71.2±7.0 mV, spike threshold −48±0.6 mV, spike amplitude 80.3±6.1 mV, and spike duration 1.58±0.24 ms; global ECoG remained isoelectric.

Methods

  • Isolated, largely ex cranio porcine brains from 6–8 month old domestic pigs were perfused 4 h postmortem for 6 h under normothermic ex vivo conditions using a custom pulsatile-perfusion system and either control perfusate or BEx perfusate; comparison groups were unperfused 10 h PMI and flushed 1 h PMI.
  • The study used approximately 300 post-mortem pig brains during technology development, with 32 independent brains undergoing BEx perfusion for the reported data; analyses included ultrasonography, micro-CT angiography, MRI, histology/immunohistochemistry, electron microscopy, patch-clamp electrophysiology, ECoG, LPS-induced cytokine/chemokine multiplex assays, and arteriovenous metabolic measurements.
  • Data are reported as means ± SEM and analyzed with two-tailed paired/unpaired t-tests, one-way ANOVA with Dunnett’s post hoc, Kruskal-Wallis with Benjamini-Krieger-Yekutieli post hoc, or Chi-square tests, with significance set at P ≤ 0.05.

Limitations

  • The paper states that no global ECoG/EEG activity reemerged, that it is uncertain whether longer perfusion could restore global activity, and that inhibitory agents in the BEx perfusate may dampen network activity; it also states that restoration of molecular/cellular processes should not be extrapolated to normal brain function or awareness.
  • The paper states that delayed cellular death starting 72 h to weeks after ischemia cannot be excluded, that younger animals may have greater resilience to anoxic injury, and that further development, optimization, longer perfusion studies, and ethical standard operating procedures are needed.
  • Evident from the text: this is an isolated ex vivo porcine brain model without integrated brain function, and several analyses rely on representative images or small independent-brain sample sizes (e.g., n=3 for some imaging/EM/electrophysiology experiments), which limits generalization.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-019-1099-1

Cites

51 of 51 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 15 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Spontaneous excitatory postsynaptic currents (sEPSCs) were present at a holding potential of −70 mV in BEx-perfused hippocampal slices.

    “we observed the presence of spontaneous excitatory postsynaptic currents (sEPSCs) at a holding potential of −70 mV”
    Quote in the paperSubstantial claim
  • Findingown data

    BEx formulation diminished caspase 3 activation, as shown by increased actCASP3 in CA1 and dentate gyrus across all control groups compared to BEx.

    “we found an increase in the percentage of nuclei positive for actCASP3 in CA1 and dentate gyrus across all control groups, indicating that the BEx formulation diminished caspase 3 activation”
    Quote in the paperSubstantial claim
  • Findingown data

    BEx-perfused brains showed robust flow through the major arteries of the brain.

    “Color Doppler analysis of BEx-perfused brains revealed robust flow through the major arteries of the brain”
    Quote in the paperSubstantial claim
  • Findingown data

    Spontaneous global electrical activity did not reemerge during BEx perfusion, as evidenced by isoelectric ECoG.

    “spontaneous global activity did not reemerge as evidence by isoelectric ECoG activity during the course of BEx perfusion”
    Quote in the paperSubstantial claim
  • Findingown data

    Reperfusion occurred throughout the brain via cannulation of the bilateral carotid arteries.

    “indicating successful reperfusion throughout the brain via cannulation of the bilateral carotid arteries”
    Quote in the paperSubstantial claim
  • It may state two separate claims as one. 62
  • The quote only partly states this claim. 8
  • Probably not a substantial claim. 48

  • It may state two separate claims as one. The quote only partly states this claim.

    Findingown data

    10h PMI brains exhibited deterioration of gray-white matter contrast, reduction in LCC and APD, and collapse of the lateral ventricles, indicative of decreased water content.

    “10h PMI brains exhibited a deterioration of gray-white matter contrast, reduction in LCC and APD, as well as collapse of the lateral ventricles”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    CP brains demonstrated pervasive tissue damage, decreased global signal intensity, ventricular expansion, increased LCC, APD, and gray-white matter contrast, indicating edema.

    “CP brains demonstrated pervasive tissue damage, decreased global signal intensity, ventricular expansion, along with increased LCC, APD, and gray-white matter contrast, indicating the presence of edema”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In prefrontal neocortex, BEx conditions preserved neuronal cell morphology and density.

    “Analysis of the prefrontal neocortex also provided evidence for preservation of both neuronal cell morphology and density under BEx conditions”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one. The quote only partly states this claim.

    Findingown data

    BEx perfusion restored glial inflammatory responses, as LPS injection increased IL-1α, IL-1β, IL-6, and IL-8 relative to other conditions.

    “Under BEx conditions, we observed an increase in interleukin (IL)-1α, IL-1β, IL-6, and IL-8 relative to other conditions”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one. The quote only partly states this claim.

    Findingown data

    The brain vasculature remained responsive to pharmacological intervention; nimodipine caused a significant and sustained increase in flow velocity under BEx.

    “We observed a significant and sustained increase in flow velocity post-intervention, indicating that the vasculature retained dilatory functionality in response to pharmacological stimulation”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    EM analyses indicated preservation of pre-synaptic vesicular structure in BEx conditions compared to 1h PMI, with substantial degradation in 10h PMI and CP brains.

    “EM analyses indicated preservation of pre-synaptic vesicular structure in BEx conditions as compared to 1h PMI, with substantial degradation in 10h PMI and CP brains”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Recorded neurons in BEx-perfused hippocampal slices responded to supra-threshold depolarizing current with repetitive action potential firing and expressed voltage-gated fast Na+ and delayed-rectifier K+ currents.

    “All recorded neurons responded to supra-threshold depolarizing current stimuli with repetitive action potential firing (Fig. 6c) and expressed both voltage-gated fast Na+ currents and delayed-rectifier K+ currents (Fig. 6d).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    The epidural temperature following the craniectomy ranged from 12-15°C.

    “The epidural temperature following the craniectomy ranged from 12–15°C.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    In 10h PMI, GAD1-positive neurons displayed contracted cytoplasmic morphology and loss of somal GAD1-positive axonal contacts.

    “these cells displayed contracted cytoplasmic morphology and a loss of somal GAD1-positive axonal contacts”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Wet-to-dry weight showed decreased water content in 10h PMI controls and increased in CP conditions, with no significant difference between 1h PMI and BEx perfusate brains.

    “we measured the wet-to-dry weight of cerebral samples (Fig. 3b), and observed a decrease in water content in 10h PMI controls and an increase in CP conditions, with no significant difference between 1h PMI and BEx perfusate brains”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Fiber bundle density was decreased under 10h PMI and CP conditions, with no difference between 1h PMI and BEx perfusion.

    “found a decrease in the bundle density under 10h PMI and CP conditions, demonstrating an overall deterioration of axonal architecture; in contrast, there was no difference in bundle density between 1h PMI and BEx perfusion”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    BEx perfusion maintained microvascular patency and dilatory functionality for 6h following a 4h LPP.

    “Overall, these data demonstrate restoration and maintenance of microvascular patency and dilatory functionality for 6h following a 4h LPP.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    At normothermia, average oxygen expenditure was approximately 2 mL/min/100 g and glucose expenditure approximately 6.5 mg/min/100 g tissue.

    “At normothermia, the average oxygen and glucose expenditure was approximately 2 mL/min/100 g and 6.5 mg/min/100 g tissue, respectively”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one. The quote only partly states this claim.

    Findingown data

    Mitochondria in BEx perfusion conditions demonstrated normal ultrastructure, whereas 1h PMI and the vast majority of 10h PMI and control-perfused specimens displayed swollen morphology with damaged cristae.

    “mitochondria in BEx perfusion conditions demonstrated normal ultrastructure”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Neuronal density was comparable between BEx and 1h PMI controls in CA1 and dentate gyrus, with reduction in 10h PMI and CP conditions.

    “Assessment of neuronal density also revealed comparable values between BEx and 1h PMI controls in both CA1 and dentate gyrus, with a reduction in 10h PMI and CP conditions”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Neuronal membrane properties and action potential parameters in BEx-perfused hippocampal slices were in accordance with previous values/literature.

    “Neuronal membrane capacitance (Cm=125±43 pF), input resistance (Rin=195±63 MΩ), membrane time constant (τm=32.3±8.4 ms), and membrane resting potential (Vm=−71.2±7.0 mV) were in accordance with previous values37.”
    • Also stated in: § Synaptic organization and neuronal activity · plain
      “the action potential parameters (spike threshold=−48±0.6 mV, spike amplitude=80.3±6.1 mV, spike duration=1.58±0.24 ms) were similar to previous literature38”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [37] Kowalski, J., Gan, J., Jonas, P. & Pernía-Andrade, A. J. Intrinsic membrane properties determine hippocampal differential firing pattern in vivo in anesthetized rats. Hippocampus 26, 668–682 (2016). https://doi.org/10.1002/hipo.22550. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [38] Simkin, D. et al. Aging-related hyperexcitability in CA3 pyramidal neurons is mediated by enhanced A-type K+ channel function and expression. J. Neurosci. 35, 13206–13218 (2015). source

      Checked before — its earlier verdict is held back until a new check runs.

  • It may state two separate claims as one.

    Findingown data

    Arteriovenous gradients demonstrated consistent consumption of glucose and oxygen as brains reached normothermia, with concurrent CO2 production and a physiological drop in pH.

    “Arteriovenous gradients demonstrated consistent consumption of glucose and oxygen as the brains reached normothermia, with a concurrent production in CO2 and a physiological drop in pH39.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Lam, T. I. et al. Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase. Proc. Natl Acad. Sci. USA 110, E4362–E4368 (2013). source

      Checked before — its earlier verdict is held back until a new check runs.

  • The quote only partly states this claim.

    Findingown data

    BEx perfusion prevents edema in the porcine brain after 4h PMI.

    “prevents edema”
    • Also stated in: § Preservation of tissue integrity · plain
      “with no significant difference between 1h PMI and BEx perfusate brains”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Oxygen consumption rates were comparable with previously reported in vivo values, but glucose consumption was higher than normal yet in line with rates reported for brains recovering from prolonged global ischemia.

    “our findings indicate that oxygen consumption rates were comparable with previously reported in vivo values55, but glucose consumption was higher than normal56 yet in line with rates reported for brains recovering from prolonged global ischemia57.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [55]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [56]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [57]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • It may state two separate claims as one.

    Findingown data

    Venous sodium and potassium concentrations normalized into physiological range after one hour of perfusion, potentially signaling restoration of active Na+/K+ homeostasis.

    “venous concentrations of both sodium and potassium normalized into physiological range after one hour of perfusion, potentially signaling the restoration of active Na+/K+ homeostasis”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In BEx perfusate conditions, oligodendrocytes demonstrate normal ultrastructure with numerous cisterns of rough endoplasmic reticulum, and mitochondria have electron-dense matrix and many cristae.

    “In BEx perfusate conditions, oligodendrocytes demonstrate normal ultrastructure with numerous cisterns of rough endoplasmic reticulum (rer), and mitochondria have electron-dense matrix and many cristae.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    BEx and 10h PMI conditions demonstrate an increase in number of axons orthogonally oriented to the pial surface, while 1h PMI and control perfusate specimens exhibit an increase in axons oriented in more acute angles.

    “BEx and 10h PMI conditions demonstrate an increase in number of axons orthogonally oriented to the pial surface, while 1h PMI and control perfusate specimens exhibit an increase in axons oriented in more acute angles.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    In the hippocampal white matter of 1h PMI, 10h PMI, and control perfusate conditions, numerous cells contain segments of destroyed cytoplasm, while oligodendrocytes have light cytoplasm with numerous vacuoles and swollen mitochondria; in 10h PMI samples, mitochondria may not be visible due to extensive destruction.

    “In the hippocampal white matter of 1h PMI, 10h PMI, and control perfusate conditions, numerous cells contain segments of destroyed cytoplasm (pink), while oligodendrocytes have light cytoplasm with numerous vacuoles and swollen mitochondria (yellow); in 10h PMI samples, mitochondria may not be visible due to extensive destruction.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Micro-computed tomography angiogram (CTA) demonstrates patency of major arterial vessels as well as arterioles.

    “Micro-computed tomography angiogram (CTA) demonstrates patency of major arterial vessels as well as arterioles (left).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Neurons exhibit a swollen morphology in 1h PMI, and significant cellular destruction in 10h PMI and control perfusate conditions, while neurons in the BEx perfusion condition display typical elongated morphology.

    “Note that neurons exhibit a swollen morphology in 1h PMI, and significant cellular destruction in 10h PMI and control perfusate conditions (arrowheads), while neurons in the BEx perfusion condition display typical elongated morphology (arrows).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingnone given

    Phase 1 is defined by a normal, low-resistance waveform.

    “Phase 1 is defined by a normal, low-resistance waveform; phase 2 displayed an increase in PSV, narrowed systole, and decreased EDV; phase 3 demonstrates decreased PSV and loss of EDV; phase 4 exhibits a reverberated “no-flow” phenomenon.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingown data

    In 1h PMI controls, certain mitochondria display normal ultrastructure and electron-dense matrix, while others exhibit a more swollen morphology.

    “In 1h PMI controls, certain mitochondria display normal ultrastructure and electron-dense matrix (m), while others exhibit a more swollen morphology (yellow).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    In 10h PMI conditions, ependymal cells demonstrate lightened cytoplasm with numerous vacuoles and swollen mitochondria; adherence junctions are preserved, but the cell membrane is destroyed in several positions.

    “Contrastingly, in 10h PMI conditions ependymal cells demonstrate lightened cytoplasm with numerous vacuoles (v) and swollen mitochondria (yellow). Although adherence junctions (aj) are preserved, the cell membrane is destroyed in several positions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    Power waveform analysis from a representative control perfusate brain during Phase 3 evidenced a MFV of 3 cm/s.

    “Power waveform analysis from a representative control perfusate brain during Phase 3 with corresponding trace (right) evidencing a MFV of 3 cm/s.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    There is evidence of clear cell destruction and the presence of enlarged vacuoles under 10h PMI and control perfusate conditions.

    “There is evidence of clear cell destruction and the presence of enlarged vacuoles under 10h PMI and control perfusate conditions (arrowheads).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Phase 3 demonstrates decreased PSV and loss of EDV.

    “Phase 1 is defined by a normal, low-resistance waveform; phase 2 displayed an increase in PSV, narrowed systole, and decreased EDV; phase 3 demonstrates decreased PSV and loss of EDV; phase 4 exhibits a reverberated “no-flow” phenomenon.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Nissl stains of the occipital neocortex demonstrate preserved regional cytoarchitectonics as well as neuronal structure in BEx perfused brains.

    “Nissl stains (top) with higher magnification of boxed area (below) of the occipital neocortex demonstrating preserved regional cytoarchitectonics as well as neuronal structure in BEx perfused brains.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    At 1h PMI there is robust nuclear localization of cleaved caspase-3 across all brain regions; however, this signal decreases with increased post-mortem intervals.

    “At 1h PMI there is robust nuclear localization of cleaved caspase-3 across all brain regions; however, this signal decreases with increased post-mortem intervals.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Pericytes and endothelial cells in hippocampal CA1 capillaries after BEx perfusion demonstrate normal ultrastructure and contain cell nuclei, mitochondria, and cisterns of rough endoplasmic reticulum; adherence junctions between endothelial cells are visible.

    “Pericytes and endothelial cells (highlighted semitransparent green and blue, respectively) demonstrate normal ultrastructure and contain cell nuclei (N), mitochondria (m), and cisterns of rough endoplasmic reticulum (rer). Adherence junctions between endothelial cells (aj) are visible.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Phase 2 displayed an increase in PSV, narrowed systole, and decreased EDV.

    “Phase 1 is defined by a normal, low-resistance waveform; phase 2 displayed an increase in PSV, narrowed systole, and decreased EDV; phase 3 demonstrates decreased PSV and loss of EDV; phase 4 exhibits a reverberated “no-flow” phenomenon.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Maximum intensity projections of Neurogranin (NRGN) show a preservation of typical morphology of cortical pyramidal neurons under BEx perfusion, with swollen morphology under 1h PMI conditions.

    “Maximum intensity projections of the excitatory neuron marker, Neurogranin (NRGN; green), show a preservation of typical morphology of cortical pyramidal neurons under BEx perfusion (arrows), with swollen morphology under 1h PMI conditions (arrow).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingown data

    An inverted pyramidal neuron structure is appreciable under BEx perfusion.

    “An inverted pyramidal neuron structure is also appreciable under BEx perfusion (arrow).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Interpretationreasoning

    The compounds and neuronal activity blockers in BEx perfusate are washed out during preparation of acute hippocampal slices, possibly explaining the discrepancy between whole cell patch-clamp recordings and global cortical activity.

    “These compounds, as well as neuronal activity blockers, are washed out during the preparation of acute hippocampal slices for electrophysiological recordings, possibly explaining the observed discrepancy between whole cell patch-clamp recordings and global cortical activity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductreasoning

    The perfusion chamber maintains a relative humidity level of approximately 95% to prevent the brain surface from drying because the brain is not bathed in fluid.

    “the chamber is temperature-controlled and maintains a relative humidity level ~95% to prevent the surface of the brain from drying, since the brain is not bathed in fluid.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductnone given

    Global electrophysiological monitoring was performed throughout the experimental timeline.

    “Global electrophysiological monitoring was performed throughout the experimental timeline.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    The surgical procedures required approximately 10 minutes to complete, subjecting the brains to that length of warm anoxia.

    “In total, these procedures required approximately 10 mins to complete, thereby subjecting brains to this length of warm anoxia.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductnone given

    In all experiments, cerebral perfusate flow was monitored and arterial pressure was adjusted to maintain relatively constant cerebral flow.

    “In all experiments, the cerebral perfusate flow was monitored and arterial pressure adjusted appropriately to maintain relatively constant cerebral flow.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductreasoning

    For reproducibility and comparisons, all ultrasonography measurements were conducted on the pericallosal artery anterior to the genu of the corpus callosum.

    “For reproducibility and comparisons all measurements were conducted on pericallosal artery anterior to the genu of corpus callosum.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Premisecitation

    The rodent brain comprises only 14% white matter, while pig and human approximate 30 and 50%, respectively.

    “the rodent brain comprising only 14% white matter, while pig and human approximate 30 and 50%, respectively61,62.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [61]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [62]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Interpretationcitation

    The increased plasticity in younger-aged brains may have contributed to the overall cellular recovery.

    “Therefore, the increased plasticity in younger-aged brains may have contributed to the overall cellular recovery.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Interpretationreasoning

    The surgical preparation transected the cranial nerves and removed the spinal nerve roots and dorsal root ganglia, which precludes potential transmission of noxious stimuli to the brain.

    “Due to the surgical preparation, the cranial nerves were transected and the spinal nerve roots and dorsal root ganglia were removed, which precludes potential transmission of noxious stimuli to the brain.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductown data

    Global electrical activity was measured during the experimental timeline via two independent methods: the BIS brain monitoring system and a 5×4 platinum-iridium electrode grid.

    “Global electrical activity was measured during the experimental timeline via two independent methods: commercially-available, clinical-grade BIS™ brain monitoring system (Medtronic) and 5×4 platinum-iridium electrode grid (AD-Tech; Racine, Wisconsin; Cat #: FG20C-SP10X-000) with an exposed surface of 2.8 mm and a 10 mm contact spacing covering the frontal-parieto-occipital convexity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Premisecitation

    Younger animals demonstrate a greater resilience to anoxic injury than adults.

    “previous work has shown that younger animals demonstrate a greater resilience to anoxic injury than adults58”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [58]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    For all staining procedures except intravascular hemoglobin fluorescence detection, tissue sections from each experimental group were co-stained on the same slide.

    “For all staining procedures except for intravascular hemoglobin fluorescence detection, tissue sections from each experimental group were co-stained on the same slide.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The microvasculature under BEx perfusion is patent and maintaining flow.

    “These findings suggest that the microvasculature under BEx perfusion is patent and maintaining flow”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    32 independent brains underwent BEx perfusion, and data from these are presented.

    “32 independent brains underwent BEx perfusion, data from which are presented herein.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Approximately 300 post-mortem pig brains were utilized from USDA-approved food production facilities during technology development and the final study.

    “approximately 300 post-mortem pig brains were utilized from USDA-approved food production facilities during the process of technology development (surgical approach, vascular isolation, device configuration, perfusate formulation and so on) and the final study described herein.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Figure 2 (a-c) ultrasonography data are from a representative brain, and the experiment was repeated in n = 15 independent brains with similar results.

    “Figure 2 (a-c) Ultrasonography data are from a representative brain; the experiment was repeated in n = 15 independent brains with similar results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one. The quote only partly states this claim.

    Study design and conductnone given

    For quantifying fiber bundle density, three representative brightfield images were taken from each biological replicate, randomized, and counted by a blinded observer.

    “For quantifying fiber bundle density, three representative (8x) brightfield images were taken from each biological replicate and randomized.”
    • Also stated in: § Histological data analysis and quantification. · plain
      “A blinded observer counted the number of fiber bundles present per image.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductnone given

    Series resistance was always compensated by 70-80% and monitored throughout the experiment.

    “Series resistance was always compensated by 70–80% and monitored throughout the experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Tissue sections were imaged using an LSM880 confocal microscope with identical settings across all experimental conditions.

    “Tissue sections were imaged utilizing an LSM880 confocal microscope (Zeiss; Jena, Germany) equipped with a motorized stage using 10x (0.3 NA) or 20x (0.8 NA) objective lenses with identical settings across all experimental conditions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Figure 4 (a) images are from a representative brain per condition, and the experiment was repeated in n=6 independent brains per condition with similar results.

    “Figure 4 (a) Images are from a representative brain per condition; the experiment was repeated in n=6 independent brains per condition with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Membrane voltage was corrected off-line for a calculated liquid junction potential of −10 mV.

    “Membrane voltage was corrected off-line for a calculated liquid junction potential of −10 mV.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The quote only partly states this claim.

    Study design and conductown data

    A total of 41 capillaries across n=3 independent brains were analyzed for the number of examined prefrontal neocortical capillaries filled with BEx perfusate.

    “A total of 41 capillaries across n=3 independent brains were analyzed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. The quote only partly states this claim.

    Study design and conductown data

    Measurements of myelinated fiber orientation were made from randomized representative images by a blinded observer.

    “Representative images were accumulated from each biological replicate from all experimental groups and randomized.”
    • Also stated in: § Histological data analysis and quantification. · boosted
      “Measurements were made by a blinded observer from the white matter boundary to layer 1, which could be clearly delineated by the presence of dense horizontal fibers.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Image is from a representative brain; the experiment was repeated in n=3 independent brains with similar results.

    “Image is from a representative brain; the experiment was repeated in n=3 independent brains with similar results”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductnone given

    A blinded observer manually counted DAPI+ objects and actCASP3+ objects using ImageJ.

    “A blinded observer manually counted DAPI+ objects and actCASP3+ objects utilizing the cell counter function in ImageJ software (NIH).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Images are from a representative brain per condition; the experiment was repeated in n=3 independent brains per condition with similar results.

    “Images are from a representative brain per condition; the experiment was repeated in n=3 independent brains per condition with similar results”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductnone given

    Images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group.

    “Images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Data are from a representative rendering; the experiment was repeated in n=3 independent brains with similar results.

    “Data are from a representative rendering; the experiment was repeated in n=3 independent brains with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductnone given

    For all cell marker stains, cells were excluded if below a minimum intensity level, exhibiting cellular vacuoles greater than the size of the nucleus, or demonstrating clear cellular destruction.

    “For all cell marker stains, cells were excluded if below a minimum intensity level, exhibiting cellular vacuoles greater than the size of the nucleus, or demonstrating clear cellular destruction.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    A total of 54 capillaries were examined across n=3 independent brains.

    “A total of 54 capillaries were examined across n=3 independent brains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Image is from a representative capillary; a total of 54 capillaries were identified across n=3 independent brains.

    “Image is from a representative capillary; a total of 54 capillaries were identified across n=3 independent brains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group with similar results.

    “Images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group with similar results.”
    • Also stated in: § Extended Data · plain
      “Images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group with similar results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    For both (a & b), images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group with similar results.

    “For both (a & b), images are from a representative brain from each condition; experiments were repeated in n=3 independent brains per group with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Traces are from representative brains; the experiment was repeated in n=3 independent brains for each condition with similar results.

    “Traces are from representative brains; the experiment was repeated in n=3 independent brains for each condition with similar results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductnone given

    Confocal tile scans of tissue sections stained for GFAP, IBA1, NRGN, and GAD1 were normalized to identical image surface area, randomized, and analyzed by a blinded observer.

    “Confocal tile scans (using the 10x or 20x objectives described above) of tissue sections stained for GFAP, IBA1, NRGN, and GAD1 were normalized to identical image surface area, randomized, and subsequently analyzed by a blinded observer for the objects of interest.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    A blinded observer processed images and manually counted objects of interest and morphology using ImageJ for cresyl violet staining.

    “A blinded observer processed the images as described above and manually counted objects of interest and morphology utilizing the cell counter function in ImageJ software (NIH).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Images are from a representative brain per condition; the experiment was repeated in n=3 independent brains with similar results.

    “Images are from a representative brain per condition; the experiment was repeated in n=3 independent brains with similar results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Image is from a representative capillary; a total of 41 capillaries were identified across n=3 independent brains.

    “Image is from a representative capillary; a total of 41 capillaries were identified across n=3 independent brains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Images are from a representative brain from each condition; experiments were repeated in n=6 independent brains per group with similar results.

    “Images are from a representative brain from each condition; experiments were repeated in n=6 independent brains per group with similar results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Traces are from a representative brain; the experiment was repeated in n=3 independent brains with similar results.

    “Traces are from a representative brain; the experiment was repeated in n=3 independent brains with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Images are from representative brains for each brain region; experiments were repeated in n=3 independent brains per group with similar results.

    “Images are from representative brains for each brain region; experiments were repeated in n=3 independent brains per group with similar results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Image adjustments were uniformly applied to all experimental conditions in Zeiss Zen.

    “Image adjustments were uniformly applied to all experimental conditions in Zeiss Zen.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Pairwise comparisons by two-tailed Chi-square analysis with Yates correction with df=1 and n=786 axons were analyzed per pairwise comparison; P-values specified within; n=3 brains per condition.

    “Pairwise comparisons by two-tailed Chi-square analysis with Yates correction with df=1 and n=786 axons were analyzed per pairwise comparison; P-values specified within; n=3 brains per condition.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. It may state two separate claims as one.

    Study design and conductown data

    Synapses in the hippocampal CA1 stratum oriens were imaged by a blind operator, and 8 randomly chosen synapses per animal per condition were analyzed by another blind observer.

    “Synapses in the hippocampal CA1 stratum oriens were imaged by a blind operator and 8 randomly chosen synapses per animal per condition (approximately 24 synapses/condition) were subjected to analysis by another blind observer.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    In comparison to 1h PMI and control perfusate conditions, fluorescent signal is found in precapillary arterioles and capillaries of both 10h PMI controls and BEx perfusate, indicating that the BEx perfusate reperfuses cerebral microcirculation.

    “In comparison to 1h PMI and control perfusate conditions, fluorescent signal is found in precapillary arterioles (arrowhead) and capillaries (arrow) of both 10h PMI controls and BEx perfusate, indicating that the BEx perfusate reperfuses cerebral microcirculation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Study design and conductown data

    n=3 measurements were taken from 3 independent brains per condition.

    “n=3 measurements taken from 3 independent brains per condition.”
    Quote in the paperSubstantial claim
83 of 83

  • The paper relies on this without support.

    Absence of global electrical activity detectable by BIS and ECoG indicates absence of remnant awareness.

    “electrical activity was also examined to confirm that the brain did not display activity consistent with remnant awareness.”
    “No such electrical activity was ever observed.”
1 of 1

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Schmidt, V. Comparative Anatomy of the Pig Brain: an Integrative Magnetic Resonance Imaging (MRI) Study of the Porcine Brain with Special Emphasis on the External Morphology of the Cerebral Cortex. Thesis, Justus-Liebig-Universität Gießen (2015).

Cited by

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Related

Metadata

Authors
Vrselja, Z., Daniele, S. G., Silbereis, J., Talpo, F., Morozov, Y. M., Sousa, A. M. M., Tanaka, B. S., Skarica, M., Pletikos, M., Kaur, N., Zhuang, Z. W., Liu, Z., Alkawadri, R., Sinusas, A. J., Latham, S. R., Waxman, S. G., & Sestan, N.
Affiliations
  • Yale University (US)
  • University of Pavia (IT)
  • Boston University (US)
  • University of Pittsburgh (US)
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2019-04-17
Volume
568
Issue
7752
Pages
336-343
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-019-1099-1
PMC
PMC6844189
OpenAlex
W2939414360
Cited by
311
References
51
Citations by year
2026: 23 · 2025: 26 · 2024: 39 · 2023: 44 · 2022: 37 · 2021: 55 · 2020: 36 · 2019: 49
FWCI
41.73
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • Revista de Psicología (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Repositorio Institucional UCA (Pontificia Universidad Católica Argentina) (submittedVersion, cc-by-nc-sa)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • National Institutes of Health (1s10od021845-01, 2r01mh113257-06, UL1TR001863)
  • National Institute of Mental Health (#R01-MH113257, RF1MH117064)
  • National Institute of General Medical Sciences (T32GM007205)
  • National Center for Advancing Translational Sciences
Topics
  • Health Sciences › Medicine › Surgery › Organ Transplantation Techniques and Outcomes
  • Health Sciences › Medicine › Clinical Neurology › Traumatic Brain Injury and Neurovascular Disturbances
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Organ Donation and Transplantation
Keywords
cerebral microcirculation, pulsatile perfusion, ischemia-reperfusion injury, cerebral edema, brain energy metabolism, synaptic activity, glial inflammation, cell death, pig brain, normothermic perfusion
MeSH terms
Animals, Autopsy, Brain, Cell Survival, Hypoxia, Brain, Cerebral Arteries, Brain Ischemia, Cerebrovascular Circulation, Disease Models, Animal, Inflammation, Microcirculation, Neuroglia, Neurons, Perfusion, Swine, Synapses, Time Factors, Vasodilation, Reperfusion Injury, Caspase 3

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-11-10, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-10 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Statistics and arithmetic are recomputed, not judged.

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