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Interpericyte tunnelling nanotubes regulate neurovascular coupling


Interpericyte tunnelling nanotubes regulate neurovascular coupling

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Signalling between cells of the neurovascular unit, or neurovascular coupling, is essential to match local blood flow with neuronal activity. Pericytes interact with endothelial cells and extend processes that wrap capillaries, covering up to 90% of their surface area1,2. Pericytes are candidates to regulate microcirculatory blood flow because they are strategically positioned along capillaries, contain contractile proteins and respond rapidly to neuronal stimulation3,4, but whether they synchronize microvascular dynamics and neurovascular coupling within a capillary network was unknown. Here we identify nanotube-like processes that connect two bona fide pericytes on separate capillary systems, forming a functional network in the mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We provide evidence that these (i) have an open-ended proximal side and a closed-ended terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii) carry organelles including mitochondria, which can travel along these processes, and (iii) serve as a conduit for intercellular Ca²⁺ waves, thus mediating communication between pericytes. Using two-photon microscope live imaging, we demonstrate that retinal pericytes rely on IP-TNTs to control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries. IP-TNT damage following ablation or ischaemia disrupts intercellular Ca²⁺ waves, impairing blood flow regulation and neurovascular coupling. Notably, pharmacological blockade of Ca²⁺ influx preserves IP-TNTs, rescues light-evoked capillary responses and restores blood flow after reperfusion. Our study thus defines IP-TNTs and characterizes their critical role in regulating neurovascular coupling in the living retina under both physiological and pathological conditions.
IP-TNTs connect pericytes on distal capillaries in a soma-to-process configuration a–c, Pericyte extending a nanotube process, which we name an IP-TNT (dotted line), across an adjacent capillary. All retinas presented IP-TNTs. d, e, IP-TNT diameter (n = 86 IP-TNTs/pericytes, n = 3 mice; two-tailed Student’s t-test, NS, not significant) and length (n = 91 IP-TNTs/pericytes, n = 3 mice; two-tailed Mann–Whitney U test, ***P < 0.001); data are mean ± s.e.m. f, Incidence of short versus long IP-TNTs (n = 91 IP-TNTs/pericytes, n = 3 mice). g, h, Labelling of IP-TNTs with phalloidin (g) and lectin (h), showing F-actin cytoskeleton. i, Fluorescein electroporated into a pericyte with IP-TNT diffuses from soma and IP-TNT into distal pericyte (pipette in green; replicated five times). j, Pericyte with IP-TNT (inset), visualized by confocal imaging, is selected for FIB-SEM. k, Montage of FIB-SEM images of the pericyte in j (inset) highlighting its nucleus (purple), IP-TNT (yellow, dotted line), distal pericyte processes (dpp, red) and capillaries (green). l, Reconstruction of FIB-SEM images in k. m, Open-ended side of IP-TNT is continuous with the pericyte cytoplasm. n, Mitochondria within IP-TNT (left) and at its end-foot (ef; right). o–q, IP-TNTs contain vesicles (o) and endoplasmic reticulum (ER; p), and membrane-to-membrane contacts exist between the IP-TNT ef (yellow) and dpp (red) (q). Four hundred FIB-SEM sections of an IP-TNT were analysed. r, Top, CX43 immunostaining (green) in two IP-TNT-connected pericytes. Bottom, inset shows CX43 labelling (green) between the IP-TNT ef and the dpp. s–u, Fluorescein electroporated in the presence of carbenoxolone accumulates in proximal pericyte and IP-TNT (s, t, left), delineating contact between IP-TNT ef (green) and dpp (red) (u). Right, higher magnification of inset (boxed in s–u). v, Higher magnification of inset (boxed in u, right) shows localization of CX43-positive plaques (yellow) at interface between IP-TNT ef (green) and dpp (red) (replicated 12 times). The same IP-TNT-coupled pericytes in s–u were processed for CX43 labelling (replicated three times). Source data
IP-TNTs are a conduit for pericyte-to-pericyte communication in living retinas a, Setup for two-photon laser scanning microscopy (TPLSM). b, In vivo imaging using intravenously applied FITC (to visualize capillaries) and intravitreally injected lectin (to label the basement membrane of pericytes) shows a network of IP-TNTs linking distal capillaries, observed in all retinas analysed. c, d, Short and long IP-TNTs exist in all vascular plexuses. e, Colabelling with phalloidin to detect F-actin (top) and lectin to visualize pericytes/IP-TNTs (bottom) shows that IP-TNTs contain an F-actin cytoskeleton. f, Time-lapse imaging shows mitochondria travelling within an IP-TNT (arrow; replicated 3 times). g, TPLSM recordings show intercellular Ca²⁺ wave (ICWs) propagating through an IP-TNT (dotted line; replicated in 57 IP-TNTs, n = 20 mice). h, ICWs are synchronous Ca²⁺ increases in IP-TNT-coupled pericyte pairs (grey). i, j, ICW frequency decreases with carbenoxolone (CBX) treatment (in j, vehicle: n = 18 capillaries, n = 4 mice; CBX: n = 16 capillaries, n = 4 mice; *P = 0.029). k, Longitudinal analysis of light-evoked changes in capillary diameter in vivo (blue, dilation, n = 39 capillaries, n = 2 mice; red, constriction, n = 30 capillaries, n = 2 mice; *P < 0.05, **P < 0.01). l, TPLSM quantification of red blood cells (RBC) per time unit after light stimulation (replicated in 224 capillaries, n = 28 mice). m, IP-TNT-linked capillaries undergo opposite blood flow responses (dilation: n = 27 capillaries, n = 2 mice; constriction: n = 10 capillaries, n = 2 mice; ***P < 0.001). n, ICWs in IP-TNT-linked capillaries before and after light stimulation (n = 13 capillaries per group, n = 5 mice per group; NS, not significant). o, p, A decrease in Ca²⁺ transient frequency correlates with dilation (pericyte 1), whereas increased frequency correlates with constriction in the coupled pericyte (pericyte 2). p shows Ca²⁺ transient frequency changes in IP-TNT-linked pericytes (n = 13 pericytes per group, n = 5 mice per group; **P < 0.01). q, r, Pericytes at constricting and dilating capillaries display increases and decreases in Ca²⁺ transient frequency, respectively (constriction: n = 10 pericytes, n = 5 mice; dilation: n = 16 pericytes, n = 5 mice, **P = 0.008). Data are mean ± s.e.m. j, m, n, two-tailed Mann–Whitney U test; k, two-tailed ANOVA Dunnett’s test; p–r, two-tailed Student’s t-test. Source data
Ischaemia damages IP-TNTs and leads to microvascular dysfunction a, Retinal ischaemia induced by transient ligature of ophthalmic vessels. b, TPLSM imaging of pericytes and capillaries at 10 and 40 min after induction of ischaemia or sham surgery (replicated 3 times per group). c, TPLSM shows reduction in capillary diameter in ischaemia relative to sham controls (sham: n = 75 capillaries, n = 3 mice; ischaemia: n = 29 capillaries, n = 3 mice; **P < 0.01, ***P < 0.001). d, Time-lapse TPLSM imaging of IP-TNT (dotted line) disintegration during ischaemia (arrows; replicated 3 times). e, Ischaemia also ruptures IP-TNTs ex vivo. f, Increase in damaged IP-TNTs in ischaemia relative to controls (ischaemia: n = 6 mice; sham-operated: n = 4; *P = 0.038). g–i, IP-TNT damage induced by ischaemia–reperfusion causes loss of light-evoked capillary dilation (blue) and constriction (red) (pre-ischaemia: dilation, n = 21 capillaries, n = 3 mice, constriction, n = 21 capillaries, n = 3 mice; post-ischaemia: dilation, n = 18 capillaries, n = 4 mice, constriction, n = 21 capillaries, n = 5 mice). h, Capillary responses are impaired in ischaemia (dilation: sham, n = 46 capillaries, n = 3 mice, ischaemia–reperfusion (I–R), n = 20, n = 4 mice; constriction: sham, n = 60, n = 3 mice, reperfusion, n = 21, n = 5 mice; *P = 0.016, **P = 0.002). i, Blood flow is compromised in ischaemia–reperfusion (dilation: sham, n = 31 capillaries, n = 5 mice, reperfusion, n = 5 capillaries, n = 5 mice; constriction: sham, n = 28 capillaries, n = 5 mice, reperfusion, n = 10 capillaries, n = 5 mice; *P = 0.034, NS, not significant). j–m, Nifedipine restores light-evoked capillary responses (dilation: sham + nifedipine, n = 14 capillaries, n = 4, reperfusion + vehicle, n = 27 capillaries, n = 5, reperfusion + nifedipine, n = 17 capillaries, n = 4; constriction: sham + nifedipine, n = 20 capillaries, n = 4, reperfusion + vehicle, n = 21 capillaries, n = 5, reperfusion + nifedipine, n = 18 capillaries, n = 4; *P < 0.05, ***P < 0.001, NS, not significant). n, Blood flow is impaired after ischaemia–reperfusion, but restored by nifedipine (dilation: sham + nifedipine, n = 16 capillaries, n = 4, reperfusion + vehicle, n = 13 capillaries, n = 5, reperfusion + nifedipine, n = 12 capillaries, n = 4; constriction: sham + nifedipine, n = 10 capillaries, n = 4, reperfusion + vehicle, n = 11 capillaries, n = 5, reperfusion + nifedipine, n = 13 capillaries, n = 4; *P < 0.05, NS, not significant). Data are mean ± s.e.m. c, m, n, two-tailed ANOVA Tukey’s test; f, h, i, two-tailed Student’s t-test. Source data
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Nature | Vol 585 | 3 September 2020 | 91
Interpericyte tunnelling nanotubes regulate
neurovascular coupling
Luis Alarcon-Martinez1,2,4 ✉, Deborah Villafranca-Baughman1,2,4, Heberto Quintero1,2,
J. Benjamin Kacerovsky3, Florence Dotigny1,2, Keith K. Murai3, Alexandre Prat1,2,
Pierre Drapeau1,2 & Adriana Di Polo1,2 ✉
Signalling between cells of the neurovascular unit, or neurovascular coupling, is
essential to match local blood ow with neuronal activity. Pericytes interact with
endothelial cells and extend processes that wrap capillaries, covering up to 90% of
their surface area1,2. Pericytes are candidates to regulate microcirculatory blood ow
because they are strategically positioned along capillaries, contain contractile
proteins and respond rapidly to neuronal stimulation3,4, but whether they
synchronize microvascular dynamics and neurovascular coupling within a capillary
network was unknown. Here we identify nanotube-like processes that connect two
bona de pericytes on separate capillary systems, forming a functional network in the
mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We
provide evidence that these (i) have an open-ended proximal side and a closed-ended
terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii)
carry organelles including mitochondria, which can travel along these processes, and
(iii) serve as a conduit for intercellular Ca2+ waves, thus mediating communication
between pericytes. Using two-photon microscope live imaging, we demonstrate that
retinal pericytes rely on IP-TNTs to control local neurovascular coupling and
coordinate light-evoked responses between adjacent capillaries. IP-TNT damage
following ablation or ischaemia disrupts intercellular Ca2+waves, impairing blood
ow regulation and neurovascular coupling. Notably, pharmacological blockade of
Ca2+ inux preserves IP-TNTs, rescues light-evoked capillary responses and restores
blood ow after reperfusion. Our study thus denes IP-TNTs and characterizes their
critical role in regulating neurovascular coupling in the living retina under both
physiological and pathological conditions.
Pericytes embedded along capillary walls regulate microcirculatory
blood flow by contracting and relaxing to induce changes in capil-
lary diameter
. This model explains capillary dynamics, but does not
account for the need to coordinate dilation and constriction in distal
capillaries to achieve fine regulation of blood supply within a local
network. Examination of retinas from mice thatexpress red fluorescent
protein under control of the NG2 (Cspg4) promoter (NG2–DsRed),
which allows the selective visualization of retinal pericytes6, revealed
fine processes connecting neighbouring capillaries (Fig.1a), remi-
niscent of intervascular bridging strands described in fixed tissue7–13.
The colocalization of DsRed and TRITC-lectin, which labels both the
pericyte’s basement membrane and endothelial cells, showed that
these processes emerged from the pericyte soma and extended to
distal capillaries (Fig.1b, c). These pericyte-derived structures were
tubular (Supplementary Video1), with an average diameter of 500nm
(Fig.1d), resembling thin tunnelling nanotubes
. They ranged in length
from 4 to 90μm, with a bimodal length distribution; we classified them
as either short or long (shorter or longer than 30μm), and found that
short processes were more prevalent than long processes (66% and
34%, respectively) (Fig.1e, f). Retinal labelling with phalloidin, which
binds F-actin, revealed that these pericyte-derived processes contain
an F-actin cytoskeleton (Fig.1g, h), a structural requirement of thin
tunnelling nanotubes.
An important phenotypic criterion of thin tunnelling nanotubes
is that they connect two distinct cells14. Analysis of retinas from
NG2–DsRed mice revealed that the pericyte elaborating each thin
tunnelling nanotube, referred to here as the proximal pericyte, con-
nected with the process of a pericyte on a distal capillary, thus termed
the distal pericyte (Extended Data Fig.1a–d). We therefore named
these processes interpericyte tunnelling nanotubes (IP-TNTs). To
investigate whether these structures generally connect two peri-
cytes, we performed single-pericyte electroporation of fluorescein, a
low-molecular-weight dye (332Da). Fluorescein diffused rapidly from
the soma of the proximal pericyte into the IP-TNT and, critically, into
Received: 12 December 2018
Accepted: 6 July 2020
Published online: 12 August 2020
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1Department of Neuroscience, University of Montreal, Montreal, Quebec, Canada. 2University of Montreal Hospital Research Centre, Montreal, Quebec, Canada. 3Centre for Research in
Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada. 4These
authors contributed equally: Luis Alarcon-Martinez, Deborah Villafranca-Baughman. e-mail:;
Content courtesy of Springer Nature, terms of use apply. Rights reserved
... Here, our work confirmed that TNTs are elastic structures that bend under radial force. Indeed, in vivo observations show that curved TNT-like structures present in brain and embryo tissue (Osswald et al., 2015;Alarcon-Martinez et al., 2020;Scheiblich et al., 2021), indicating that they tolerate radial forces derived from adjacent cells and extracellular matrix. Furthermore, we measured the spring constant of TNTs (79.1 ± 16.2 pN/μm). ...
Full-text available
Tunneling nanotubes (TNTs) are thin membrane tubular structures that interconnect physically separated cells. Growing evidence indicates that TNTs play unique roles in various diseases by facilitating intercellular transfer of signaling and organelles, suggesting TNTs as a potential target for disease treatment. The efficiency of TNT-dependent communication is largely determined by the number of TNTs between cells. Though TNTs are physically fragile structures, the mechanical properties of TNTs and the determinants of their mechanical stability are still unclear. Here, using atomic force microscope (AFM) and microfluidic techniques, we investigated the mechanical behavior and abundance of TNTs in human embryonic kidney (HEK293) cells upon the application of forces. AFM measurements demonstrate that TNTs are elastic structures with an apparent spring constant of 79.1 ± 16.2 pN/μm. The stiffness and membrane tension of TNTs increase by length. TNTs that elongate slower than 0.5 μm/min display higher mechanical stability, due to the growth rate of F-actin inside TNTs being limited at 0.26 μm/min. Importantly, by disturbing the cytoskeleton, membrane, or adhesion proteins of TNTs, we found that F-actin and cadherin connection dominantly determines the tensile strength and flexural strength of TNTs respectively. It may provide new clues for screening TNT-interfering drugs that alter the stability of TNTs.
... Eyes were immediately collected, post-fixed in PFA, and processed to generate cryosections or retinal flat mounts as previously described (Pernet et al., 2007;Almasieh et al., 2011Almasieh et al., , 2013. Retinas were incubated in the following primary antibodies overnight at 4 C overnight (cross-sections) or 3 days (whole-mount retinas): RBPMS (0.25 mg/mL, PhosphoSolutions, Aurora, CO), Disc1 (0.8 mg/mL, Santa Cruz Biotechnology, Dallas, TX), Miro1 (2 mg/mL, Invitrogen, Rockford, IL), GFP (2 mg/mL, Invitrogen), or RFP (1 mg/mL, Invitrogen), followed by fluorophore-conjugated secondary antibodies (2-4 mg/mL, Invitrogen Two-photon microscopy live imaging of mitochondrial transport Live imaging of mitochondrial transport in RGC axons was performed by TPLSM as described (Alarcon-Martinez et al., 2020. Mice were anesthetized and placed on a customized platform with controlled temperature (37 C) and air ventilation. ...
Full-text available
Deficits in mitochondrial transport are a common feature of neurodegenerative diseases. We investigated whether loss of components of the mitochondrial transport machinery impinge directly on metabolic stress, neuronal death, and circuit dysfunction. Using multiphoton microscope live imaging, we showed that ocular hypertension, a major risk factor in glaucoma, disrupts mitochondria anterograde axonal transport leading to energy decline in vulnerable neurons. Gene- and protein-expression analysis revealed loss of the adaptor disrupted in schizophrenia 1 (Disc1) in retinal neurons subjected to high intraocular pressure. Disc1 gene delivery was sufficient to rescue anterograde transport and replenish axonal mitochondria. A genetically encoded ATP sensor combined with longitudinal live imaging showed that Disc1 supplementation increased ATP production in stressed neurons. Disc1 gene therapy promotes neuronal survival, reverses abnormal single-cell calcium dynamics, and restores visual responses. Our study demonstrates that enhancing anterograde mitochondrial transport is an effective strategy to alleviate metabolic stress and neurodegeneration.
... Co-staining of the eyes with anti-CD39 antibody and different vascular markers demonstrated the presence of CD39 on all components of the vessel wall, including CD31 + /IB4 + vascular endothelial cells which share their basement membranes with adjacent NG2 + /Phalloidin + pericytes, and also contractile SMA-α + /Phalloidin + smooth muscle cells (SMC) wrapped in a circumferential pattern around larger arterioles ( Fig. 2a and Fig. S1). Interestingly, the close-up view of the deep and intermediate plexuses of the mouse retina validated recent data on the presence of so-called "interpericyte tunnelling nanotubes" that connect two bona fide pericytes on separate capillary systems and regulate neurovascular coupling in the living retina [41], and further extend these observations by showing that these fine structures do not express CD39 and as a consequence are unable to metabolize ATP (Fig. 2a, inset). CD39 is also expressed, albeit faintly compared to blood vessels, on other ocular structures, including rhodopsin + OS of photoreceptor cells (Fig. 1b), NeuN + neuronal cell bodies located in the ganglion cell layer (GCL), as well as P2Y 12 R + microglial cells, which mainly reside in two synaptic compartments of the neural parenchyma: the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and in the optic nerve head (Fig. 2b). ...
Ocular ATP and adenosine have emerged as important signalling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging in combination with in situ enzyme histochemistry, flow cytometric analysis and single cell transcriptomics to characterize the pattern of purine metabolism in the mouse and human eyes. This study identified NTPDase1/CD39 and ecto-5’-nucleotidase/CD73 as major ecto-nucleotidases, which are selectively expressed in the optic nerve head, vascular endothelial and perivascular cells, outer segments of photoreceptors, retinal microglia, and cornea and coordinately control ATP and adenosine levels. The relevance of the CD73-adenosine axis was confirmed by flash electroretinography showing that pharmacological inhibition of CD73 in dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes and a loss of retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and characterizes its critical role in protecting the retina from light-induced phototoxicity.
... 2PCI has been used to gain mechanistic insights into both of these phases. Acutely after stroke, 2PCI has been used to record the time course and spatial extent of neuronal dysfunction 178,179 , to test the roles of specific receptors in the propagation of cortical spreading depressions 180,181 and to quantify changes in [Ca 2+ ] i handling in non-neuronal cells such as astrocytes 178,180,182 , microglia 183,184 and pericytes 185 . In the weeks following stroke, 2PCI in the peri-infarct cortex has been used to investigate how sensory circuits change throughout recovery 186,187 . ...
In vivo two-photon calcium imaging (2PCI) is a technique used for recording neuronal activity in the intact brain. It is based on the principle that, when neurons fire action potentials, intracellular calcium levels rise, which can be detected using fluorescent molecules that bind to calcium. This Primer is designed for scientists who are considering embarking on experiments with 2PCI. We provide the reader with a background on the basic concepts behind calcium imaging and on the reasons why 2PCI is an increasingly powerful and versatile technique in neuroscience. The Primer explains the different steps involved in experiments with 2PCI, provides examples of what ideal preparations should look like and explains how data are analysed. We also discuss some of the current limitations of the technique, and the types of solutions to circumvent them. Finally, we conclude by anticipating what the future of 2PCI might look like, emphasizing some of the analysis pipelines that are being developed and international efforts for data sharing. Two-photon calcium imaging is a technique used for recording neuronal activity in the brain. In this Primer, Grienberger et al. outline the experimental design and execution of two-photon calcium imaging, providing examples of ideal preparations and how data are analysed.
... These compartments exhibit different responses during neurovascular coupling, participating in the complex interpretation of the functional response measured by mesoscopic imaging, such as the blood-oxygen-level-dependent (BOLD) signal for functional magnetic resonance imaging (fMRI) 13 or the Power Doppler signal for functional ultrasound 14 . These different signatures can be interpreted using optical imaging providing information on neurovascular coupling at the microscopic scale, but within a limited field of view 1,[15][16][17] . Such local interpretation fails to account for the large-scale information on the global vascular system architecture to which they belong. ...
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The advent of neuroimaging has increased our understanding of brain function. While most brain-wide functional imaging modalities exploit neurovascular coupling to map brain activity at millimeter resolutions, the recording of functional responses at microscopic scale in mammals remains the privilege of invasive electrophysiological or optical approaches, but is mostly restricted to either the cortical surface or the vicinity of implanted sensors. Ultrasound localization microscopy (ULM) has achieved transcranial imaging of cerebrovascular flow, up to micrometre scales, by localizing intravenously injected microbubbles; however, the long acquisition time required to detect microbubbles within microscopic vessels has so far restricted ULM application mainly to microvasculature structural imaging. Here we show how ULM can be modified to quantify functional hyperemia dynamically during brain activation reaching a 6.5-µm spatial and 1-s temporal resolution in deep regions of the rat brain. Functional ultrasound localization microscopy monitors cerebrovascular blood flow by detecting the flow of injected microbubbles, providing access to brain activity at high spatiotemporal resolution.
Ageing, which can be defined as functional decline of an organism with time, is a major factor of susceptibility to various chronic diseases, which constitute a huge burden for health care systems worldwide. The study of the mechanisms encompassing the alterations associated with age-related phenotypes has been a matter of great interest, however, due to its complexity, they are far from being elucidated. Despite the fact that ageing relies on a myriad of distinct responses, several hallmarks of ageing have been established, which include, among others altered intercellular communication. In this review, we aim at providing a comprehensive and critical perspective on the impact and role of intercellular communication in ageing. Although intercellular communication during ageing can assume different forms we will focus on: 1) the canonical senescence associated secretory phenotype, 2) direct cell-cell communication through gap junctions or tubule-like structures and 3) long distance communication, involving extracellular vesicles and paracrine communication mediated by Connexin-containing hemichannels. Furthermore, we will discuss whether changes in intercellular communication can be considered a cause or consequence of ageing.
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Diabetic retinopathy (DR) is the most common complication of diabetes mellitus (DM), which can lead to visual impairment and even blindness in severe cases. DR is generally considered to be a microvascular disease but its pathogenesis is still unclear. A large body of evidence shows that the development of DR is not determined by a single factor but rather by multiple related mechanisms that lead to different degrees of retinal damage in DR patients. Therefore, this article briefly reviews the pathophysiological changes in DR, and discusses the occurrence and development of DR resulting from different factors such as oxidative stress, inflammation, neovascularization, neurodegeneration, the neurovascular unit, and gut microbiota, to provide a theoretical reference for the development of new DR treatment strategies.
Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions spanning lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we found that Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting the formation and lengths of TNTs. Upon Arp2/3 inhibition, epidermal growth factor receptor kinase substrate 8 (Eps8) exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 resulting in increased TNTs. In these conditions, Eps8 interaction with proteins enhancing filament turnover and depolymerization were reduced. Our data suggest a shift in the equilibrium (and usage of common actin proteins players) between branched and linear actin polymerization to form different cell protrusions.
Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.Significance StatementDevelopmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis (XLRS), a well-defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature and glia. In mice lacking RS1, we found an array of novel phenotypes which present around eye-opening, are linked to glutamatergic neurotransmission, and affect visual processing. These data identify novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.
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Intercellular communication is a fundamental property of multicellular organisms, necessary for their adequate responses to changing environment. Tunneling nanotubes (TNTs) represent a novel means of intercellular communication being a long cell-to-cell conduit. TNTs are actively formed under a broad range of stresses and are also proposed to exist under physiological conditions. Development is a physiological condition of particular interest, as it requires fine coordination. Here we discuss whether protrusions shown to exist during embryonic development of different species could be TNTs or if they represent other types of cell structure, like cytonemes or intercellular bridges, that are suggested to play an important role in development.
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Increasing evidence indicates that pericytes are vulnerable cells, playing pathophysiological roles in various neurodegenerative processes. Microvascular pericytes contract during cerebral and coronary ischemia and do not relax after re-opening of the occluded artery, causing incomplete reperfusion. However, the cellular mechanisms underlying ischemia-induced pericyte contraction, its delayed emergence, and whether it is pharmacologically reversible are unclear. Here, we investigate i) whether ischemia-induced pericyte contractions are mediated by alpha-smooth muscle actin (α-SMA), ii) the sources of calcium rise in ischemic pericytes, and iii) if peri-microvascular glycogen can support pericyte metabolism during ischemia. Thus, we examined pericyte contractility in response to retinal ischemia both in vivo, using adaptive optics scanning light ophthalmoscopy and, ex vivo, using an unbiased stereological approach. We found that microvascular constrictions were associated with increased calcium in pericytes as detected by a genetically encoded calcium indicator (NG2-GCaMP6) or a fluoroprobe (Fluo-4). Knocking down α-SMA expression with RNA interference or fixing F-actin with phalloidin or calcium antagonist amlodipine prevented constrictions, suggesting that constrictions resulted from calcium- and α-SMA-mediated pericyte contractions. Carbenoxolone or a Cx43-selective peptide blocker also reduced calcium rise, consistent with involvement of gap junction-mediated mechanisms in addition to voltage-gated calcium channels. Pericyte calcium increase and capillary constrictions became significant after 1 h of ischemia and were coincident with depletion of peri-microvascular glycogen, suggesting that glucose derived from glycogen granules could support pericyte metabolism and delay ischemia-induced microvascular dysfunction. Indeed, capillary constrictions emerged earlier when glycogen breakdown was pharmacologically inhibited. Constrictions persisted despite recanalization but were reversible with pericyte-relaxant adenosine administered during recanalization. Our study demonstrates that retinal ischemia, a common cause of blindness, induces α-SMA- and calcium-mediated persistent pericyte contraction, which can be delayed by glucose driven from peri-microvascular glycogen. These findings clarify the contractile nature of capillary pericytes and identify a novel metabolic collaboration between peri-microvascular end-feet and pericytes.
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The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.
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Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at -20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling.
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Blood flow into the brain is dynamically regulated to satisfy the changing metabolic requirements of neurons, but how this is accomplished has remained unclear. Here we demonstrate a central role for capillary endothelial cells in sensing neural activity and communicating it to upstream arterioles in the form of an electrical vasodilatory signal. We further demonstrate that this signal is initiated by extracellular K⁺ —a byproduct of neural activity—which activates capillary endothelial cell inward-rectifier K⁺ (KIR2.1) channels to produce a rapidly propagating retrograde hyperpolarization that causes upstream arteriolar dilation, increasing blood flow into the capillary bed. Our results establish brain capillaries as an active sensory web that converts changes in external K⁺ into rapid, 'inside-out' electrical signaling to direct blood flow to active brain regions.
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Pericytes, spatially isolated contractile cells on capillaries, have been reported to control cerebral blood flow physiologically, and to limit blood flow after ischaemia by constricting capillaries and then dying. Paradoxically, a recent paper dismisses the idea of pericytes controlling cerebral blood flow, despite confirming earlier data showing a role for pericytes. We show that these discrepancies are apparent rather than real, and depend on the new paper defining pericytes differently from previous reports. An objective definition of different sub-classes of pericyte along the capillary bed is needed to develop novel therapeutic approaches for stroke and disorders caused by pericyte malfunction. Pubmed:
The central role of the cardiovascular system is to maintain adequate capillary perfusion. The spatially and temporally heterogeneous nature of capillary perfusion has been reported in some organs. However, such heterogeneous perfusion properties have not been sufficiently explored in the retina. Arguably, spatial and temporal heterogeneity of capillary perfusion could be more predominant in the retina than that in other organs. This is because the retina is one of the highest metabolic demand neural tissues yet it has a limited blood supply due to optical requirements. In addition, the unique heterogeneous distribution of retinal neural cells within different layers and regions, and the significant heterogeneity of intraretinal oxygen distribution and consumption add to the complexity. Retinal blood flow distribution must match consumption of nutrients such as oxygen and glucose within the retina at the cellular level in order to effectively maintain cell survival and function. Sophisticated local blood flow control in the microcirculation is likely required to control the retinal capillary perfusion to supply local retinal tissue and accommodate temporal and spatial variations in metabolic supply and demand. The authors would like to update the knowledge of the retinal microvessel and capillary network and retinal oxidative metabolism from their own studies and the work of others. The coupling between blood supply and energy demands in the retina is particularly interesting. We will mostly describe information regarding the retinal microvessel network and retinal oxidative metabolism relevant to the spatial and temporal heterogeneity of capillary perfusion. We believe that there is significant and necessary spatial and temporal heterogeneity and active regulation of retinal blood flow in the retina, particularly in the macular region. Recently, retinal optical coherence tomography angiography (OCTA) has been widely used in ophthalmology, both experimentally and clinically. OCTA could be a valuable tool for examining retinal microvessel and capillary network structurally and has potential for determining retinal capillary perfusion and its control. We have demonstrated spatial and temporal heterogeneity of capillary perfusion in the retina both experimentally and clinically. We have also found close relationships between the smallest arterioles and capillaries within paired arterioles and venules and determined the distribution of smooth muscle cell contraction proteins in these vessels. Spatial and temporal heterogeneity of retinal capillary perfusion could be a useful parameter to determine retinal microvessel regulatory capability as an early assay for retinal vascular diseases. This topic will be of great interest, not only for the eye but also other organs. The retina could be the best model for such investigations. Unlike cerebral vessels, retinal vessels can be seen even at the capillary level. The purpose of this manuscript is to share our current understanding with the readers and encourage more researchers and clinicians to investigate this field. We begin by reviewing the general principles of microcirculation properties and the spatial and temporal heterogeneity of the capillary perfusion in other organs, before considering the special requirements of the retina. The local heterogeneity of oxygen supply and demand in the retina and the need to have a limited and well-regulated retinal circulation to preserve the transparency of the retina is discussed. We then consider how such a delicate balance of metabolic supply and consumption is achieved. Finally we discuss how new imaging methodologies such as optical coherence tomography angiography may be able to detect the presence of spatial and temporal heterogeneity of capillary perfusion in a clinical setting. We also provide some new information of the control role of very small arterioles in the modulation of retinal capillary perfusion which could be an interesting topic for further investigation.
Photothrombosis of blood vessels refers to the activation of a circulating photosensitive dye with a green light to induce clotting in vivo (Watson et al., 1985). Previous studies have described how a focused green laser could be used to noninvasively occlude pial arterioles and venules at the brain surface (Schaffer et al., 2006; Nishimura et al., 2007; Shih et al., 2013). Here we show that small regions of the capillary bed can similarly be occluded to study the ischemic response within the capillary system of the mouse cerebral cortex. The advantage of this approach is that the ischemic zone is restricted to a diameter of approximately 150-250 μm. This permits higher quality two-photon imaging of degenerative processes that would be otherwise difficult to visualize with models of large-scale stroke, due to excessive photon scattering. A consequence of capillary occlusion is leakage of the blood-brain barrier (BBB). Here, through the use of two-photon imaging data sets, we show how to quantify capillary leakage by determining the spatial extent and localization of intravenous dye extravasation.
Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal M?ller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100?, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP(+) astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. ? 2017 Wiley Periodicals, Inc.
Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.