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.
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All data sets and/or analyses generated in this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank HbO2 Therapeutics, specifically J. Baqai, G. Dube, and R. Buskar, for providing the Hemopure product and their input in optimizing oxygen delivery methods; C. Aardema, D. Faulkner and R. Robertson for technical assistance and discussions on the perfusion system; T. Constable and M. Qiu for help with MRI imaging; A. Liu and T. Wing for assisting in ECoG discussions and data acquisition; and various members of our laboratory community for their comments on the manuscript. We thank A. Duque for assistance in using the equipment from the Yale Macaque Brain Resource, supported by NIH MH113257. This work was supported by the NIH BRAIN Initiative MH117064, NIH shared instrument grant OD021845, which funded purchase of the microSPECT/CT scanner, and NIH/NIGMS Medical Scientist Training Grant T32GM007205.
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Nature thanks Insoo Hyun, Costantino Iadecola, Jonathan Moreno and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Authors and Affiliations
Contributions
Z.V. and S.G.D contributed equally to this work. Z.V., S.G.D., J.S. and N.S. designed the BEx technology and the research described herein. Z.V. and S.G.D. developed the surgical procedure, performed the perfusion experiments, and collected and processed tissue samples for subsequent analyses. Z.V. and S.G.D. performed the MRI studies and analysed the data. Y.M.M. conducted the EM analysis. Z.V., S.G.D., J. S., N.S. and Y.M.M. analysed the EM data. F.T. and B.S.T. performed electrophysiology studies. F.T., B.S.T. and S.G.W. analysed the electrophysiology data. Z.V., S.G.D., Z.W.Z., Z.L. and A.J.S. performed the CTA studies and analysed the data. S.G.D., J.S., A.M.M.S., M.S., M.P. and N.K. conducted histological analyses. Z.V., S.G.D., J.S. and M.S. analysed and quantified the histological data. R.A. performed the ECoG studies and analysed the data. S.R.L. contributed to the bioethical aspects of the research. N.S. conceived and supervised the project. Z.V., S.G.D. and N.S. wrote the manuscript and prepared figures. All authors discussed and commented on the data.
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Competing interests
Z.V., S.G.D., J.S. and N.S. have disclosed these findings to the Yale Office of Cooperative Research, which has filed a patent to ensure broad use of the technology. All protocols, methods, perfusate formulations, and components of the BrainEx technology remain freely available for academic and non-profit research. While the Hemopure product was provided in accordance to a material transfer agreement between HbO2 Therapeutics and Yale University via N.S., the Company had no influence on the study design or interpretation of the results. No author has a financial stake in, or receives compensation from, HbO2 Therapeutics. All other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Detailed schematic of the perfusion circuit.
Complete blueprint of the perfusion circuitry and system. Individual components are listed on the right and in Supplementary Table 3.
Extended Data Fig. 2 Schematic representation of surgical brain and vascular isolation procedure with connection to the perfusion system.
a, Process for initially reducing the porcine skull after decapitation at the C3 vertebra. Dotted orange lines represent bone cuts through the nasofrontal suture, extending ventrally through the mandible (1); through the zygomatic process of the temporal bone and malar bone (2, 3); and through the supraorbital process (4). Dotted maroon lines represent disarticulation of the temporomandibular and atlanto-occipital joints. BA, basilar artery; VA, vertebral artery; OA, occipital artery; CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; RM, rete mirabile; OphA, ophthalmic artery. Smaller vessels such as the ramus anastomoticus, arteria anastomotica, and variable branches originating from the ascending pharyngeal artery are not shown. b, Arterial dissection, ligation, and removal of cranial soft tissue. To prevent vascular shunting, the vertebral, basilar, occipital, external carotid and ophthalmic arteries were ligated with sutures and/or cauterized along with the ramus anastomoticus, arteria anastomotica, and smaller branches originating from the ascending pharyngeal artery, while the internal carotid arteries were left patent. c, Neurocranial opening and reduction. Dark orange dots represent burr holes that are connected with dashed lines; orange shaded area represents the area of the skull that is entirely removed at the end of the procedure. d, Mainly ex cranio whole brain connected to the BEx device. e, Components used for the connection of the porcine brain to the arterial ends of the BEx system.
Extended Data Fig. 3 Perfusion dynamics.
a, Ultrasound traces of the waveform cycle (systole and diastole) from each of the four phases observed under CP conditions. Each phase occurs in chronological order during the experimental timeline, revealing progressive deterioration of the low-resistance pressure waveform structure. In each phase, there is a corresponding decrease in the relative mean flow velocity (MFV), culminating in negligible net forward flow (reverberated pattern) in phase 4 (6 h LOP). Phase 1 is defined by a normal, low-resistance waveform; phase 2 displays an increase in peak systolic velocity (PSV), narrowed systole, and decreased end diastolic velocity (EDV); phase 3 demonstrates decreased PSV and loss of EDV; phase 4 exhibits a reverberated ‘no-flow’ phenomenon. Intervening in phase 3 with an increase in mean arterial pressure resulted in a transient increase in flow (red trace versus pink trace) that reverted back to negligible forward flow soon after in phase 4 (red and dark blue trace versus pink and sky blue). Traces are from a representative brain; the experiment was repeated in n = 3 independent brains with similar results. b, Power waveform analysis from a representative CP brain during phase 3 with corresponding trace (right) showing an MFV of 3 cm s–1. c, Power waveform analysis from a representative CP brain at 6 h LOP demonstrating phase 4 dynamics with corresponding trace (right) depicting MFV of 0 cm s–1. By contrast, power waveform analysis from a representative BEx brain (below) taken at 6 h LOP shows phase 1 dynamics. b, c, Traces are from representative brains; the experiment was repeated in n = 3 independent brains for each condition with similar results. d, Resistance index (RI) during the course of experimentation in control and BEx perfusate conditions. RI was calculated using Doppler ultrasound measurement and the formula RI = (PSV – EDV)/PSV. Intervention period is included above CP curve to delineate the time period in which mean arterial pressures were increased during CP conditions. n = 3 measurements taken from 3 independent brains per condition. e, RI at normothermia under CP and BEx conditions. Two-tailed unpaired t-test (t = 5.638, d.f. = 28) for mean values obtained during normothermic conditions; n = 15 collapsed measurements across n = 3 brains per condition while the brains were at normothermia (hours 2–6). Mean ± s.e.m.
Extended Data Fig. 4 Serial coronal sections of 3D renderings of micro-CTA.
Each box depicts a maximum intensity projection of a 1-cm coronal section at the plane indicated in the top left corner. Micro-CTA demonstrates patency of major arterial vessels as well as arterioles (left). Corresponding gross anatomical structures along with arterial supply areas were traced and mapped onto each micro-CTA rendering (right). Bottom right, abbreviations for anatomical structures and vessels. Scale bar, 1 cm. Data are from a representative rendering; the experiment was repeated in n = 3 independent brains with similar results.
Extended Data Fig. 5 Restoration and maintenance of microcirculation and capillary integrity.
a, 3D rendering of high-resolution specimen CTA of the hippocampus of BEx-perfused brain revealing extensive vascular network (left). Scale bar, 1 mm. Middle, enlarged area of the red box with three examples (numbered red lines) of pre-capillary arterioles of varying size. Scale bar, 50 μm. Right, relative scale of microcirculatory vessels with the corresponding vessel diameters. The technical limit of the high-resolution specimen-CTA is 25 μm. Image is from a representative brain; the experiment was repeated in n = 3 independent brains with similar results. b, Intravascular haemoglobin fluorescence in the CA1 field across all experimental conditions. In comparison to 1 h PMI and CP conditions, fluorescent signal is found in precapillary arterioles (arrowhead) and capillaries (arrows) of both 10 h PMI control and BEx brains, indicating that the BEx perfusate reperfuses cerebral microcirculation. Images are from a representative brain per condition; the experiment was repeated in n = 3 independent brains per condition with similar results c, Representative capillary electron microphotograph from the hippocampal CA1 zone after BEx perfusion. Blood vessel lumen (bl) is filled with an acellular, electron-dense material (asterisk), which represents the haemoglobin-based BEx perfusate. Pericytes and endothelial cells (semitransparent green and blue, respectively) show normal ultrastructure and contain cell nuclei (N), mitochondria (m), and cisternae of rough endoplasmic reticulum (rer). Adherens junctions (aj) between endothelial cells are visible. Scale bar, 1 μm. Image is from a representative capillary; a total of 54 capillaries were identified across n = 3 independent brains. d, Quantification of examined capillaries that were filled with the BEx perfusate shows that most capillaries were patent and perfused. A total of 54 capillaries were examined across n = 3 independent brains. Mean ± s.e.m.
Extended Data Fig. 6 Restoration of microvascular reperfusion in the prefrontal neocortex.
a, Intravascular haemoglobin fluorescence signal in the prefrontal neocortex across all conditions. In comparison to 1 h PMI and CP conditions, the fluorescent signal is found in precapillary arterioles (arrowheads) and capillaries (arrows) of both 10 h PMI controls and BEx brains. Images are from a representative brain per condition; the experiment was repeated in n = 3 independent brains with similar results. b, Capillary electron microphotographs from the prefrontal neocortex after BEx perfusion. Blood vessel lumen is filled with an acellular, electron-dense material, which represents the haemoglobin-based BEx perfusate. Image is from a representative capillary; a total of 41 capillaries were identified across n = 3 independent brains. c, Quantification of the number of examined prefrontal neocortical capillaries filled with BEx perfusate. A total of 41 capillaries across n = 3 independent brains were analysed.
Extended Data Fig. 7 Microvascular reperfusion and cytoarchitectonics in the occipital neocortex and cerebellar cortex.
a, Micro-CTA demonstrates patency of major arterial vessels and arterioles in the occipital lobe and cerebellum. OL, occipital lobe; V, vermis; LC, lateral cortex; CP, choroid plexus; L, left; R, right. b, c, Intravascular haemoglobin fluorescent signal in the occipital neocortex (b) and cerebellar cortex (c), demonstrating signal in precapillary arterioles (arrowheads) and capillaries (arrows) in both 10 h PMI controls and BEx brains. Images are from a representative brain from each condition; experiments were repeated in n = 3 independent brains per group. d, Nissl stains of the occipital neocortex with higher magnification of boxed area below, showing preserved regional cytoarchitectonics and neuronal structure in BEx brains. Pyramidal-shaped cell bodies as well as apical and basal dendrites are present in both 1 h PMI and BEx brains. An inverted pyramidal neuron structure is also appreciable under BEx perfusion (arrow). Scale bars, 350 μm (top), 100 μm (bottom). Images are from a representative brain from each condition; experiments were repeated in n = 3 independent brains per group. e, Representative Nissl stains of the cerebellar cortex reveal preserved Purkinje cell structure (arrows) in 1 h PMI and BEx brains, when compared to 10 h PMI and CP conditions. Images are from a representative brain from each condition; experiments were repeated in n = 3 independent brains per group.
Extended Data Fig. 8 Analysis of cytoarchitectural integrity, neuronal morphology and density in the neocortex.
a, Nissl stains of prefrontal neocortex with higher magnification of boxed area below, demonstrating preserved neuronal structure and anatomical cytoarchitecture in BEx brains. Scale bars, 350 μm (top), 100 μm (bottom). Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. b, Nissl stains of primary motor cortex reveal preserved Betz cell structure (arrows) under BEx perfusion conditions, despite these cells having been axotomized following decapitation. Despite similar brain region analysis, question marks denote uncertainty of Betz cell identity due to stark cellular disintegration. Images are from a representative brain from each condition; experiments were repeated in n = 3 independent brains per group with similar results. c, Confocal tile scans of immunohistochemical stains for NeuN (green) in the prefrontal neocortex. Scale bar, 50 μm. Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. d, Maximum intensity confocal projections of NeuN staining. Neurons exhibit a swollen morphology in 1 h PMI brains, and significant cellular destruction under 10 h PMI and CP conditions (arrowheads), while neurons in the BEx condition display typical elongated morphology (arrows). Scale bar, 50 μm. Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. e, Maximum intensity projections of NRGN staining (green) show preservation of typical morphology of cortical pyramidal neurons under BEx perfusion (arrows), with swollen morphology under 1 h PMI conditions (red arrow). There is evidence of clear cell destruction and the presence of enlarged vacuoles under the 10 h PMI and CP conditions (arrowheads). Scale bar, 50 μm. Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. f, Maximum intensity projections of GAD1 staining (red). In 10 h PMI and CP specimens, GAD1 staining reveals contracted cell bodies (arrows) with a loss of GAD1-positive somal contacts as compared to 1 h PMI and BEx brains. Scale bar, 50 μm. Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. g, Number of neuronal cells present in the prefrontal neocortex. Data computed from Nissl stains. One-way ANOVA (P < 0.001, F[3,20] = 224.6) with post-hoc Dunnett’s adjustment; n = 6 brains per condition; NS, not significant. h, Percentage of cells that exhibit a swollen, ellipsoid morphology. Data analysed from Nissl stains. One-way ANOVA (P < 0.001, F[3,20] = 16.33) with post-hoc Dunnett’s adjustment; n = 6 brains per group. Mean ± s.e.m. i, j, Total numbers of NRGN+ and GAD1+ cells, respectively, in the neocortex. One-way ANOVA (NRGN+: P = 0.002, F[3,20] = 7.018; GAD1+: P < 0.001, F[3,20] = 9.153) with post-hoc Dunnett’s adjustment. n = 6 brains per group. Mean ± s.e.m.
Extended Data Fig. 9 Ultrastructure of ependymal cells and hippocampal CA1 white matter.
a, Representative electron microphotographs of hippocampal ependymal cells. In 1 h PMI controls, some mitochondria have a normal ultrastructure and electron-dense matrix (m), while others exhibit a more swollen morphology (yellow). By contrast, in 10 h PMI conditions ependymal cells have a lightened cytoplasm with numerous vacuoles (v) and swollen mitochondria (yellow). Although adherens junctions (aj) are preserved, the cell membrane is destroyed in several places. Similarly, under CP conditions, some moderately damaged but visibly intact cells make contact with entirely destroyed adjacent cells (pink), indicating destruction of the continuum of the ependymal layer. However, most of the ependymal cells from BEx-perfused brains show ultrastructural characteristics of viable cells, such as continuity of the cell membrane that covers cilia (c) and produces filopodia (f), tight junctions between adjacent cells, and intact mitochondria with electron dense matrix. In pictomicrographs with boxed areas (top), the region is enlarged in high power (below). b, Under 1 h PMI, 10 h PMI, and CP conditions, numerous cells in the hippocampal white matter contain segments of destroyed cytoplasm (pink), while oligodendrocytes have light cytoplasm with numerous vacuoles and swollen mitochondria (yellow). In 10 h PMI samples, mitochondria may not be visible, owing to extensive destruction. Under BEx perfusate conditions, most oligodendrocytes have normal ultrastructure with numerous cisterns of rough endoplasmic reticulum (rer), and mitochondria have electron-dense matrix and many cristae. Overall, myelinated axons (MA) show similar morphology across all experimental groups. Boxed areas (top) are depicted in corresponding high-power images (below). N, cell nucleus. Scale bars, 1 μm. All images are from a representative brain from each condition; experiments were repeated in n = 3 independent brains per group with similar results.
Extended Data Fig. 10 Dynamics of caspase 3 activation in the perfused and unperfused brain.
a, Confocal maximum intensity projections of immunofluorescent stainings for actCASP3 (green) in the prefrontal cortex. Boxed areas are enlarged below. Scale bars, 50 μm (top); 10 μm (bottom). Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. b, Quantification of normalized actCASP3-positive nuclei. One-way ANOVA (P < 0.001, F[3,20] = 82.3) with post-hoc Dunnett’s adjustment. n = 6 brains per condition. Mean ± s.e.m. c, Time-course analysis of actCASP3 localization in the unperfused (10 h PMI) brain at various PMIs in the CA1 field, dentate gyrus, and prefrontal neocortex. At 1 h PMI there is robust nuclear localization actCASP3 across all brain regions; however, this signal decreases with increasing PMIs. Images are from representative brains for each brain region; experiments were repeated in n = 3 independent brains per group with similar results.
Extended Data Fig. 11 Orientation and fibre bundle density of myelinated neocortical axons.
a, Immunohistochemical staining for MBP in the prefrontal neocortex (top) with high-magnification images depicting fibre orientation and bundles (bottom). Scale bars, 100 μm (top); 50 μm (bottom). Images are from a representative brain from each condition; experiments were repeated in n = 6 independent brains per group with similar results. b, Analysis of individual axonal angles in relation to the pial surface across all experimental conditions. The BEx and 10 h PMI brains show an increase in the number of axons orthogonally oriented to the pial surface, whereas the 1 h PMI and CP specimens exhibit an increase in axons oriented at more acute angles. Pairwise comparisons by two-tailed χ2 analysis with Yates correction with d.f. = 1, n = 786 axons were analysed per pairwise comparison; n = 3 brains per condition. BEx versus 1 h PMI: χ2 = 6.403 for 0–30°, χ2 = 4.341 for 30–60°, χ2 = 18.09 for 60–90°; BEx versus CP: χ2 = 4.341 for 30–60°, χ2 = 11.39 for 60–90°. NS, not significant. c, Density of myelinated fibre bundles across experimental conditions. One-way ANOVA (P < 0.001, F[3,20] = 10.78) with post-hoc Dunnett’s adjustment; n = 6 animals per group. Mean ± s.e.m.
Extended Data Fig. 12 Glial cell structure in the hippocampus.
a, b, Confocal maximum intensity projections of immunohistochemical stains for astrocytes (GFAP; red) and microglia (IBA-1; green) with DAPI (blue) counterstain in the CA1 (a) and dentate gyrus (b) regions of the hippocampus show preservation of glial cell structure under BEx perfusion conditions. As compared to 1 h PMI controls, glial cells in BEx samples demonstrate a more reactive morphology with thickened cellular processes. Scale bar, 50 μm. Images are from a representative brain from each condition; experiments were repeated in n = 3 independent brains per group with similar results.
Supplementary information
Supplementary Information
This file contains the Supplementary Discussion.
Supplementary Table
This file contains Supplementary Table 1: Components of the Control Perfusate.
Supplementary Table
This file contains Supplementary Table 2: Components of the BEx Perfusate.
Supplementary Table
This file contains Supplementary Table 3: Components of the BEx Perfusion System.
Supplementary Table
This file contains Supplementary Table 4: Components of the Exchange Solution.
Video 1: Angiography of the BEx perfused brain.
Maximum intensity projections of global computed tomography angiography of the porcine brain at normothermia during BEx perfusion. Sequential planes in the axial (left), sagittal (middle), and coronal views are displayed. Video is from a representative brain; the experiment was repeated in n=3 independent brains with similar results.
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Vrselja, Z., Daniele, S.G., Silbereis, J. et al. Restoration of brain circulation and cellular functions hours post-mortem. Nature 568, 336–343 (2019). https://doi.org/10.1038/s41586-019-1099-1
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DOI: https://doi.org/10.1038/s41586-019-1099-1
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