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  • David Attwell - Pericytes in Neurovascular Coupling in Health and Disease

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    Brain blood flow is regulated to ensure adequate power for neuronal computation. Blood flow is increased to areas where neurons are active, and this increase underlies non-invasive brain imaging using BOLD fMRI. Blood flow is controlled at the arteriole level by smooth muscle, but there is controversy over whether it is also regulated by pericytes at the capillary level. I will demonstrate that neuronal activity and the excitatory neurotransmitter glutamate evoke an outward membrane current in pericytes and dilate capillaries, and that this dilation is caused by active relaxation of pericytes rather than a passive expansion of capillaries downstream of arteriole relaxation. Glutamate-evoked dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress synthesis of the vasoconstrictor 20-HETE. In vivo, when sensory input to the neocortex releases glutamate and increases blood flow, capillaries dilate first, followed by arterioles. Capillary dilation is estimated to generate ~89% of the increase of blood flow occurring. Capillaries also have a role in pathology. Ischaemia was previously shown to lead to a contraction of pericytes. I will report that this is followed by their death, which is expected to produce an irreversible constriction of capillaries. Pericyte death is reduced by block of glutamate receptors or calcium removal, and increases with reperfusion, but is not blocked by scavenging of reactive oxygen species. These data establish, for the first time, pericytes as major regulators of cerebral blood flow, and initiators of the BOLD fMRI response. They also focus attention on prevention of pericyte death as a therapeutic strategy to reduce the long-lasting blood flow decrease which contributes to neuronal death after stroke.

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    David Attwell - Pericytes in neurovascular coupling in health and disease

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    Brain blood flow is regulated to ensure adequate power for neuronal computation. Blood flow is increased to areas where neurons are active, and this increase underlies non-invasive brain imaging using BOLD fMRI. Blood flow is controlled at the arteriole level by smooth muscle, but there is controversy over whether it is also regulated by pericytes at the capillary level. I will demonstrate that neuronal activity and the excitatory neurotransmitter glutamate evoke an outward membrane current in pericytes and dilate capillaries, and that this dilation is caused by active relaxation of pericytes rather than a passive expansion of capillaries downstream of arteriole relaxation. Glutamate-evoked dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress synthesis of the vasoconstrictor 20-HETE. In vivo, when sensory input to the neocortex releases glutamate and increases blood flow, capillaries dilate first, followed by arterioles. Capillary dilation is estimated to generate ~89% of the increase of blood flow occurring. Capillaries also have a role in pathology. Ischaemia was previously shown to lead to a contraction of pericytes. I will report that this is followed by their death, which is expected to produce an irreversible constriction of capillaries. Pericyte death is reduced by block of glutamate receptors or calcium removal, and increases with reperfusion, but is not blocked by scavenging of reactive oxygen species. These data establish, for the first time, pericytes as major regulators of cerebral blood flow, and initiators of the BOLD fMRI response. They also focus attention on prevention of pericyte death as a therapeutic strategy to reduce the long-lasting blood flow decrease which contributes to neuronal death after stroke.

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    2K views39 pages

    David Attwell - Pericytes in Neurovascular Coupling in Health and Disease

    Uploaded by

    Fondation Singer-Polignac
    Brain blood flow is regulated to ensure adequate power for neuronal computation. Blood flow is increased to areas where neurons are active, and this increase underlies non-invasive brain imaging using BOLD fMRI. Blood flow is controlled at the arteriole level by smooth muscle, but there is controversy over whether it is also regulated by pericytes at the capillary level. I will demonstrate that neuronal activity and the excitatory neurotransmitter glutamate evoke an outward membrane current in pericytes and dilate capillaries, and that this dilation is caused by active relaxation of pericytes rather than a passive expansion of capillaries downstream of arteriole relaxation. Glutamate-evoked dilation is mediated by prostaglandin E2, but requires nitric oxide release to suppress synthesis of the vasoconstrictor 20-HETE. In vivo, when sensory input to the neocortex releases glutamate and increases blood flow, capillaries dilate first, followed by arterioles. Capillary dilation is estimated to generate ~89% of the increase of blood flow occurring. Capillaries also have a role in pathology. Ischaemia was previously shown to lead to a contraction of pericytes. I will report that this is followed by their death, which is expected to produce an irreversible constriction of capillaries. Pericyte death is reduced by block of glutamate receptors or calcium removal, and increases with reperfusion, but is not blocked by scavenging of reactive oxygen species. These data establish, for the first time, pericytes as major regulators of cerebral blood flow, and initiators of the BOLD fMRI response. They also focus attention on prevention of pericyte death as a therapeutic strategy to reduce the long-lasting blood flow decrease which contributes to neuronal death after stroke.

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    Regulation of brain energy supply: where does the action begin.and end?

    David Attwell Department of Neuroscience, Physiology & Pharmacology University College London

    Overview
    Locus of control of brain energy supply arterioles versus capillaries Pericytes can dilate capillaries How fast can this work? Relevance to BOLD fMRI Effect of ischaemia on capillaries Role of pericytes in loss of control of brain energy supply in pathology

    Regulation of brain energy supply by arteriole smooth muscle vs pericytes


    smooth muscle
    endothelial cells

    pericytes neurons

    1873

    blood flow

    capillary

    350 300

    PubMed: pericyte

    Papers/year

    250 200 150 100 50 0


    1940 1950 1960 1970 1980 1990 2000 2010

    PubMed: pericyte in title Charles Marie Benjamin Rouget (1824-1904)


    Year

    Regulation of brain energy supply by arteriole smooth muscle vs pericytes


    smooth muscle
    endothelial cells

    pericytes neurons

    blood flow

    Pericytes appear designed to produce a spatially localized constriction 65% of noradrenergic innervation of vasculature is of capillaries, not arterioles

    capillary

    10 m Cblm
    SM

    Cerebellum

    Retina

    s Retina 5 m p

    p 5 m

    10 m

    Regulation of capillary diameter by noradrenaline and glutamate superfused onto cerebellar slices

    2.5 M Noradrenaline 120 100 500 M Glutamate

    Diameter (%)

    80 60 40 20 0 0 400 800

    Time (s)

    How fast do capillaries dilate in response to neuronal activity in vivo? Relevant to understanding BOLD fMRI

    Relevance to BOLD fMRI


    FLOW Hb blood vessels stellate O2 HbO2

    Purkinje basket

    granule

    Golgi

    input climbing fibre output

    input mossy fibres

    Relevance to BOLD fMRI


    FLOW Hb blood vessels stellate O2 HbO2

    Purkinje basket

    granule

    Golgi

    input climbing fibre output

    input mossy fibres

    Deoxyhaemoglobin decreases magnetic resonance signals


    Deoxy-Hb is paramagnetic Its magnetic field makes the total magnetic field less homogenous That dephases the precession of protons in the external field (T2* decreased) That in turn decreases the signal detected So more deoxy-Hb means less signal BUT..

    FLOW Hb blood vessels stellate O2 HbO2

    glutamate

    Purkinje basket

    granule

    Golgi

    input climbing fibre output

    input mossy fibres

    VOL FLOW Hb blood vessels stellate O2 HbO2

    glutamate

    Purkinje basket

    granule

    Golgi

    input climbing fibre output

    input mossy fibres

    VOL FLOW Hb blood vessels stellate O2 HbO2

    glutamate

    Purkinje basket

    granule

    Golgi

    input climbing fibre output

    input mossy fibres

    Deoxyhaemoglobin decreases magnetic resonance signals


    Deoxy-Hb is paramagnetic Its magnetic field makes the total magnetic field less homogenous That dephases the precession of protons in the external field (T2* decreased) That in turn decreases the signal detected So more deoxy-Hb means less signal BUT..neurons increase blood flow, and decrease [deoxy-Hb] so the fMRI signal increases

    BOLD imaging

    Amygdala response to fear stimulus

    Hariri et al. (2002) Science 297, 400

    Vasoactive mediators will reach pericytes first


    Neurons are ~8.4 microns from capillaries but 70 microns from arterioles

    A PIAL SURFACE 3. Pericyte dilation may spread to arteriole 0th order penetrating arteriole Key neuron astrocyte pericyte 3rd order capillary 2nd

    1st order capillary

    1. Active cells

    GLU
    B AA
    O2 K m 350M

    order capillary
    O2 K m 10M M

    2. Dilators released by active neurons and astrocytes reach pericytes before arterioles

    PgE2 20-HETE

    smooth muscle

    EP4R

    dilation constriction

    NO

    NO blocks production of constrictor 20-HETE

    How do vasodilatory messengers affect pericytes?


    Membrane current changes evoked in pericytes by glutamate and neuronal APs

    Neuronal activity evokes capillary dilation (stimulation of cerebellar parallel fibres)

    Neuronal activity dilates capillaries in brain slices what about in vivo?

    Which vessels respond first in vivo?


    2 photon imaging in vivo

    1st order capillary


    smooth muscle
    endothelial cells blood flow

    pericytes neurons

    capillary

    Collaboration with Bodil Gesslein and Martin Lauritzen, Copenhagen

    Capillaries dilate in vivo

    Capillaries dilate before arterioles in vivo (whisker pad stimulation)

    Capillaries dilate before arterioles in vivo (whisker pad stimulation)

    Capillaries dilate before arterioles in vivo (whisker pad stimulation)

    Capillaries dilate before arterioles in vivo (whisker pad stimulation)

    There are more, and larger, responses near pericytes

    Summary of data

    How important are capillary dilations?

    Calculated resistance of (i) pia - layer 4 arteriole (ii) layer 4 arteriole to venule 200um away (iii) venule pia Ratios were 0.1:0.4:0.2 so capillaries have most resistance Inserting our measured dilations suggests capillaries generate 84% of the increase of CBF (whether or not they relax actively)

    Capillaries contribute to CBF increases in two ways


    A PIAL SURFACE 3. Pericyte dilation may spread to arteriole 0th order penetrating arteriole Key neuron astrocyte pericyte 3rd order capillary 2nd order capillary B
    O2 K m 10M

    1st order capillary

    1. Active cells

    2. Dilators released by active neurons and astrocytes reach pericytes before arterioles

    (1) Capillaries dilateAA first and pericyte arterioles PgE2hyperpolarization EP4R may spread to dilation (2) The dilation evoked in dominates the decrease of resistance to flow in capillaries M the cortex O2 K m 20-HETE constriction NO
    350M

    smooth muscle

    Overview
    Locus of control of brain energy supply arterioles versus capillaries - both Pericytes can dilate capillaries - via PgE2 How fast can this work? - 1 sec Relevance to BOLD fMRI - pericytes initiate the BOLD signal

    Effect of ischaemia on capillaries Role of pericytes in loss of control of brain energy supply in pathology

    Capillaries become occluded after ischaemia

    Con

    White areas reflect areas with no perfusion following 5 mins ischaemia

    Following ischaemia, blood flow returns to arterioles but some capillaries fail to fill on reperfusion

    Isch

    Ames et al. (1968) Am J Pathol 52, 437

    The pericyte response to ischaemia


    a control ischaemia b
    Ischaemia Diameter (%) 100 50 Anoxic 0 Depolarisation 0 t = 432s 200 400 600

    10m
    t = 12s

    Time (s)

    Ca2+
    process around capillary 3Na+

    Ca2+
    myofilaments

    ATP

    3Na+ 2K+

    Ca2+

    Peppiatt, Howarth, Mobbs & Attwell (2006), Nature 443, 700

    The CBF response to ischaemia

    The CBF response to ischaemia

    -20

    20

    40

    60

    80

    100 120 mins

    Ischaemia produces a long-lasting decrease of CBF, produced by a decrease of vasodilation (in fronto-parietal cortex of gerbil)
    Leffler et al., 1989; Nelson et al., 1992; Hauck et al., 2004 Why is the constriction and reduction of blood flow so long-lasting?

    Ischaemia kills pericytes easily


    Pericytes (% dead)

    p < 0.0001

    Brain slices, Nicola Hamilton, unpublished Also occurs in vivo after MCAO: Brad Sutherland & Clare Reynell, unpublished

    100 80 60 40 20 0 Control Ischaemia

    60 min ischaemia (no glucose, no O2, iodoacetate, antimycin) followed by fixation

    Ca2+ constricted process round capillary death 3Na+

    Ca2+
    myofilaments

    ATP

    3Na+ 2K+

    Ca2+ Pericytes may die in rigor, producing a long lasting constriction Why do they die?

    Glutamate release and reperfusion promote pericyte death


    White matter ischaemia with glycolysis and oxidative phosphorylation blocked 100 100
    (% dead) Pericytes dead of pericytes %
    p = 0.006 p = 0.003 p = 0.74

    Grey matter oxygen & glucose deprivation with different times of reperfusion
    Control Control OGD for 60 min then reoxygenation OGD (60 mins) with reperfusion

    100 100

    (% dead) dead of pericytes %Pericytes

    80 80 60 60 40 40 20 20 0 0
    o ntr o C l I sc i em a h a X TT R PA M A ck blo R DA M N c blo k

    80 80 60 60 40 40 20 20 0 0

    Con OGD Con OGD 60 120

    Con OGD Con OGD 180 240

    1 hour 2 hours 3 hours Time (min)

    NMDAR blockers: 50 M AP5 + MK801 + 7-CK AMPA/KA R blockers: 25 M NBQX 1 M TTX

    reperfusion time 3 hours reperfusion increases OGD-evoked death by 60% - but a lot occurs without reperfusion!

    Nicola Hamilton, Catherine Hall & Clare Reynell, unpublished

    Glutamate release and reperfusion promote pericyte death

    Nicola Hamilton, Catherine Hall & Clare Reynell, unpublished

    MCAO evokes pericyte death in vivo

    Pericytes die at a time when endothelial cells are unaffected

    Collaboration with Brad Sutherland and Alastair Buchan, in Oxford

    The CBF response to ischaemia

    }
    -20 0 20 40 60 80

    could reflect death of pericytes

    100 120 mins

    Ischaemia produces a long-lasting decrease of CBF, produced by a decrease of vasodilation (in fronto-parietal cortex of gerbil)
    Leffler et al., 1989; Nelson et al., 1992; Hauck et al., 2004 Pericytes are a therapeutic target in stroke

    Overview
    Locus of control of brain energy supply arterioles versus capillaries - both Pericytes can dilate capillaries - via PgE2 How fast can this work? - 1 sec Relevance to BOLD fMRI - pericytes initiate the BOLD signal Effect of ischaemia on capillaries - pericytes constrict and die Role of pericytes in loss of control of brain energy supply in pathology - crucial, + for BBB

    Collaborators
    LONDON

    Claire Peppiatt

    Clare Howarth

    Peter Mobbs

    Catherine Hall

    Clare Reynell

    Niki Hamilton

    Anusha Mishra

    Fergus OFarrell

    COPENHAGEN

    OXFORD
    Bodil Gesslein Martin Lauritzen Brad Sutherland Alastair Buchan

    Regulation of brain energy supply: where does the action begin.and end?

    At capillaries!

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