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Neurovascular dysfunction in glaucoma

September 2023
Progress in Retinal and Eye Research 97(Pt 3):101217

September 2023
97(Pt 3):101217

DOI:10.1016/j.preteyeres.2023.101217

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Authors:

Luis Alarcon-Martinez

Centre for Eye Research Australia

Yukihiro Shiga

Université de Montréal

Deborah Villafranca Baughman

Université de Montréal

Jorge Cueva-Vargas

National Eye Institute

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Progress in Retinal and Eye Research 97 (2023) 101217

Available online 30 September 2023

nc-nd/4.0/).

Neurovascular dysfunction in glaucoma

Luis Alarcon-Martinez

, Yukihiro Shiga

, Deborah Villafranca-Baughman

Jorge L. Cueva Vargas

, Isaac A. Vidal Paredes

, Heberto Quintero

, Brad Fortune

Helen Danesh-Meyer

, Adriana Di Polo

Department of Neuroscience, Universit´

e de Montr´

eal, PO Box 6128, Station centre-ville, Montreal, QC, Canada

Neuroscience Division, Centre de recherche du Centre Hospitalier de l’Universit´

e de Montr´

eal (CRCHUM), 900 Saint Denis Street, Montreal, QC, Canada

Centre for Eye Research Australia, University of Melbourne, Melbourne, Australia

Discoveries in Sight Research Laboratories, Devers Eye Institute and Legacy Research Institute, Legacy Healthy, Portland, OR, USA

Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, New Zealand

ARTICLE INFO

Keywords:

Retinal ganglion cells

Glaucoma

Neurovascular unit

Functional hyperemia

Pericytes

Inter-pericyte tunneling nanotubes

Blood-brain/retinal-barriers

ABSTRACT

Retinal ganglion cells, the neurons that die in glaucoma, are endowed with a high metabolism requiring optimal

provision of oxygen and nutrients to sustain their activity. The timely regulation of blood ow is, therefore,

essential to supply ring neurons in active areas with the oxygen and glucose they need for energy. Many

glaucoma patients suffer from vascular decits including reduced blood ow, impaired autoregulation, neuro-

vascular coupling dysfunction, and blood-retina/brain-barrier breakdown. These processes are tightly regulated

by a community of cells known as the neurovascular unit comprising neurons, endothelial cells, pericytes, Müller

cells, astrocytes, and microglia. In this review, the neurovascular unit takes center stage as we examine the

ability of its members to regulate neurovascular interactions and how their function might be altered during

glaucomatous stress. Pericytes receive special attention based on recent data demonstrating their key role in the

regulation of neurovascular coupling in physiological and pathological conditions. Of particular interest is the

discovery and characterization of tunneling nanotubes, thin actin-based conduits that connect distal pericytes,

which play essential roles in the complex spatial and temporal distribution of blood within the retinal capillary

network. We discuss cellular and molecular mechanisms of neurovascular interactions and their pathophysio-

logical implications, while highlighting opportunities to develop strategies for vascular protection and regen-

eration to improve functional outcomes in glaucoma.

1. Introduction

Glaucoma is a spectrum of neurodegenerative diseases characterized

by progressive optic nerve damage that can lead to irreversible blind-

ness. Due to its age dependence and scarcity of effective treatments, the

prevalence of glaucoma is predicted to grow over the next decades

(Tham et al., 2014). A common feature in the pathophysiology of all

glaucomas is the loss of retinal ganglion cells (RGCs), the neurons that

convey visual information from the retina to the brain. The special

cytoarchitecture of RGCs with elaborate dendrites in the retina, long

projecting axons, and synaptic terminals in brain centers, places a

considerable metabolic burden on these neurons (Almasieh et al., 2012;

Wareham et al., 2022; Tribble et al., 2023). RGCs have an exceedingly

high metabolism (Casson et al., 2020) and, as such, require a precise

regulation of blood supply to meet their oxygen and nutrient demand.

For decades, it has been thought that insufcient blood ow contributes

to RGC dysfunction and subsequent neurodegeneration in glaucoma

(Flammer et al., 1999). Prior to death, RGCs enter periods of dysfunction

(Calkins, 2012; Fry et al., 2018) associated with metabolic stress (Carelli

et al., 2009; Crish et al., 2010; Inman and Harun-Or-Rashid, 2017; Ito

and Di Polo, 2017; Osborne et al., 2016; Takihara et al., 2015; Williams

et al., 2017b; Kimball et al., 2018, Quintero et al., 2022), which can be

propelled by vascular decits. It is well known that many glaucoma

patients suffer from neurovascular dysfunction including decreased

blood ow in the retina and optic nerve, reduced vessel caliber, and

capillary defects (Flammer et al., 2002; Resch et al., 2009; Wareham and

Calkins, 2020; Shiga et al., 2016, 2018). Vascular autoregulation and

light-induced neurovascular responses are severely compromised in this

disease (Garh¨

ofer et al., 2004; Grunwald et al., 1984a; Gugleta et al.,

* Corresponding author. Department of Neuroscience Universit´

e de Montr´

eal Centre de recherche du centre hospitalier de l’Universit´

e de Montr´

eal (CRCHUM), 900

rue Saint-Denis, Montreal, QC, H2X 0A9, Canada.

E-mail address: adriana.di.polo@umontreal.ca (A. Di Polo).

Contents lists available at ScienceDirect

Progress in Retinal and Eye Research

journal homepage: www.elsevier.com/locate/preteyeres

https://doi.org/10.1016/j.preteyeres.2023.101217

Received 24 July 2023; Received in revised form 23 September 2023; Accepted 25 September 2023

Progress in Retinal and Eye Research 97 (2023) 101217

2012; Kiyota et al., 2017). Notwithstanding, the mechanisms underlying

vascular dysfunction in glaucoma and their impact on RGC and optic

nerve damage are poorly understood.

Vascular abnormalities are anticipated to promote neuronal

dysfunction, hence there has been a growing interest in exploring the

contribution of neurovascular decits to glaucomatous neuro-

degeneration. In this review, we aim to provide an updated account of

clinical observations on vascular impairment in glaucoma patients as

well as recent research on the role of neurovascular cells including

pericytes, which have been a recent focus of attention in this area.

Indeed, the discovery of tunneling nanotubes connecting pericytes in the

retina and their critical role in the regulation of coordinated neuro-

vascular interactions has had major implications in our understanding of

vascular decits during glaucomatous stress (Alarcon-Martinez et al.,

2020, 2022). We also discuss evidence of vascular barrier breakdown in

glaucoma and the potential cellular and molecular mechanisms involved

in these alterations.

2. Vascular impairment in glaucoma patients

2.1. Anatomy of the inner retina and optic nerve vascular systems

The delivery of oxygen and nutrients to the retina of higher mammals

is accomplished by two separate systems: the choroid and the retinal

vasculature, which derive from branches of the ophthalmic artery. The

choroidal circulation, which supplies the retinal pigment epithelium and

photoreceptors, is one of the most vascularized structures in mammals

required to meet the high metabolic demand of the outer retina (Bill,

1985; Kur et al., 2012). The inner retina, on the other hand, is nourished

by the retinal vasculature, which is supplied by the central retinal artery

(Singh and Dass, 1960a,b). The central retinal artery typically branches

into four radial arterioles extending outward from the optic nerve head

(ONH), each distributing blood to one quadrant of the retina. The retinal

arterioles give rise to three capillary beds: i) the supercial plexus that

supplies the ganglion cell and nerve ber layers, ii) the intermediate

plexus, which irrigates the inner plexiform layer, and iii) the deep plexus

Abbreviations

Ang2 Angiopoietin-2

ATP Adenosine Triphosphate

-SMA Alpha Smooth Muscle Actin

BRB Blood-Retina-Barrier

BBB Blood-Brain-Barrier

cGMP Cyclic Guanosine Monophosphate

CX43 Connexin-43

CSF1R Colony-Stimulating Factor 1 Receptor

CX3CR1 C-X3-C Motif Chemokine Receptor

EETs Epoxyeicosatrienoic Acids

Prostaglandin E2 receptor 4

ERG Electroretinogram

Endothelin Receptor A

Endothelin Receptor B

ET-1 Endothelin-1

ET-2: Endothelin-2

ET-3: Endothelin-3

20-HETE 20-Hydroxy-Eicosatetraenoic Acid

ICWs Intercellular Ca

Waves

IOP Intraocular Pressure

IP-TNTs Interpericyte Tunneling Nanotubes

IP3Rs Inositol 1,4,5-Trisphosphate Receptors

ATP

ATP-Sensitive K

channel

Inward-Rectier K

channel

LSFG Laser Speckle Flowgraphy

MMP-9 Metalloproteinase-9

NO Nitric Oxide

NOS NO Synthase

NVC Neurovascular Coupling

NVU Neurovascular Unit

ONH Optic Nerve Head

PANX1 Purine Channel Pannexin 1

PDGF-B: Platelet-Derived Growth Factor B

PDGFR-β: PDGF receptor-β

PECAM-1 Platelet Endothelial Cell Adhesion Molecule-1

PGE

Prostaglandin E2

P2RY12 P2Y12 Purinergic Receptor

RGCs Retinal Ganglion Cells

ROS Reactive Oxygen Species

TGF-β: Transforming Growth Factor-β

TRP Transient Potential Channels

VE-cadherin Vascular Endothelial-Cadherin

VEGF-A: Vascular Endothelial Growth Factor-A

VDCC Voltage-Dependent Ca

channel

ZO-1 Zona Occludens-1

Fig. 1. Retina and optic nerve head vascular organization. (A) Schematic representation of the distinct vascular plexuses in the retina: supercial, intermediate, and

deep. (B) Organization of the vasculature in the optic nerve head region. CRA: central retinal artery; CRV: central retinal vein; PCA: posterior ciliary artery. Created

with BioRender.com.

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

that nourishes the outer plexiform layer (Newman, 2013; Korneld and

Newman, 2014) (Fig. 1A). In the human retina, the radial peripapillary

capillary plexus comprises a fourth, anterior capillary bed specialized to

serve the thickest portion of the retinal nerve ber layer (Henkind, 1967;

Scoles et al., 2009). Recent evidence from analysis of optical coherence

tomography angiography scans indicates that the thickest part of the

nasal parafoveal rim tissue is also endowed with a fourth, anterior layer

of capillaries in addition to the three widely recognized layers described

above (Hirano et al., 2018). The blood is returned to the central retinal

vein in the ONH and ultimately drained into the cavernous sinus (Harris

and Rhoton, 1976). Unlike the choroid, which is regulated by both

sympathetic and parasympathetic nerves (Alm, 1977; Kawarai and Koss,

1998; Nilsson, 1996), the retinal vasculature lacks autonomic innerva-

tion and is controlled by mechanisms whose actions are collectively

known as autoregulation (Laties, 1967; Ye et al., 1990), which is dis-

cussed below in more detail.

The ONH is considered to be critically important in glaucoma

pathogenesis, perhaps even the primary site of injury to RGC axons

(Minckler and Bunt, 1977; Quigley and Addicks, 1981; Nickells et al.,

2012; Wilkison et al., 2021). Anatomically, the ONH comprises the

prelaminar, laminar, and retrolaminar regions (Hayreh, 1997; Mack-

enzie and Ciof, 2008). Blood supply to the ONH is highly anastomotic,

deriving in large part from the short posterior ciliary arteries, which

penetrate the peripapillary sclera to feed a dense network of ramied

capillaries often via a complete or partial arteriolar circle of Zinn-Haller

including those within the lamina cribrosa (Ciof Ga, 1996). The su-

percial nerve ber layer receives blood from the central retinal artery

and the retrolaminar region also from pial arterioles (Fig. 1B). The

lamina cribrosa, a trabeculated structure of connective tissue that pro-

vides structural and metabolic support to RGC axons, is supplied by

branches from the short posterior ciliary arteries through the circle of

Zinn-Haller. Vessels in this area are exposed to a highly dynamic

biomechanical environment of varying load and strain, and a

trans-laminar pressure gradient established by the difference between

the intraocular pressure (IOP) and the cerebrospinal uid pressure

manifest in the retrobulbar optic nerve via the subarachnoid space of its

sheath (Jonas et al., 2015). Indeed, biomechanical strains and stress in

this area have been proposed to contribute to both direct and indirect

mechanisms of axon injury, including vascular decits during glau-

comatous disease pathogenesis (Sigal and Ethier, 2009; Burgoyne, 2011;

Downs, 2015).

2.2. Vessel caliber and basal blood ow alterations

It is well established that glaucoma patients suffer from vascular

decits that manifest as reduced vessel caliber, decreased blood ow in

the retina and ONH, and capillary defects. This topic has been previously

covered in excellent reviews (Flammer et al., 2002; Newman et al.,

2018; Wareham and Calkins, 2020), therefore only a summary of recent

ndings will be discussed in this section. Advances in blood ow mea-

surements and imaging technologies, including optical coherence to-

mography angiography, have provided the ability to quantify

microvascular changes in glaucoma patients alongside structural alter-

ations (Rao et al., 2020; Shin et al., 2022; Shiga et al., 2023). A number

of studies have demonstrated reduced vessel density in the ONH and in

the peripapillary region in primary open-angle glaucoma (Jia et al.,

2012, 2014; Liu et al., 2015; Yarmohammadi et al., 2016) as well as

low-tension glaucoma patients (Maekawa et al., 2014; Yabana et al.,

2017). Vessel density in the macular region and deep layers of the

parapapillary choroid were shown to be decreased in glaucomatous eyes

(Suh et al., 2016; Lee et al., 2017b,c; Rao et al., 2016, 2017; Wan et al.,

2018; Moghimi et al., 2018). At the level of retinal capillaries, neuro-

vascular coupling has been proposed to be vascular plexus-dependent

(Hormel et al., 2021). Recent imaging data using optical coherence to-

mography show that the capillary density in the supercial vascular

complex, which comprises the nerve ber layer plexus and the ganglion

cell layer plexus, is reduced in glaucoma patients (Liu et al., 2019).

Plexus-specic responses may reect differences in the metabolic de-

mand of cells, intrinsic vascular properties, and levels of vasoactive

metabolites at each location. However, the extent to which

plexus-specic light-evoked capillary responses are involved in glau-

coma pathogenesis remains an open question.

Blood ow measurements provide a readout of the functional status

of the vascular system. Quantitative studies in open-angle and low-

tension glaucoma patients demonstrated signicant blood ow alter-

ations. For example, early-stage open-angle glaucoma patients demon-

strated a reduction of retinal and ONH microvascular content relative to

normal controls (Jia et al., 2012; Liu et al., 2015; Kiyota et al., 2021). In

low-tension glaucoma patients, a reduction in ONH blood ow observed

at the earliest stages of the pathology was found to be a risk factor for

disease progression (Shiga et al., 2016, 2018). Although robust longi-

tudinal human studies are still lacking, the overall consensus from

cross-sectional studies is that there is a strong correlation between the

loss of microvasculature and blood ow and the severity of glaucoma

(Liu et al., 2015; Takusagawa et al., 2017; Shin et al., 2017; Mansoori

et al., 2017; Geyman et al., 2017; Shiga et al., 2016, 2018; Rao et al.,

2020; Kiyota et al., 2021). While basal blood ow alterations are

important, the next sections argue that decits in the dynamic ability of

the system to regulate blood supply in response to neuronal activity

plays an even more critical role in functional visual decits associated

with glaucoma.

2.3. Vascular autoregulation decits

In healthy individuals, vascular beds in certain tissues of the body

exhibit an intrinsic ability to maintain constant blood ow over a large

range of perfusion pressures and varying metabolic demand, a process

known as autoregulation. In the retina and ONH, vascular autor-

egulation refers to the capacity of these tissues to sustain a stable blood

supply despite changes in ocular perfusion pressure (Prada et al., 2016).

The ocular perfusion pressure is the force driving blood through the

intraocular vasculature, estimated as the difference between arterial

blood pressure and IOP under the assumption that the intraocular

venous pressure is close to the IOP (Attariwala et al., 1994; Costa et al.,

2014; Glucksberg and Dunn, 1993; Guidoboni et al., 2014). In glaucoma

patients, the central retinal vein pressure can be higher than the IOP,

which may lead to lower perfusion pressure in the retina and ONH, ul-

timately contributing to disease worsening (Pillunat et al., 2014). The

autoregulation capacity of the system is conventionally assessed by

repeated blood ow measurements, obtained before and after an in-

crease or decrease in ocular perfusion pressure, which reect the slower

time course of effects known as static autoregulation (Prada et al.,

2016). This component is part of the myogenic response, which refers to

the intrinsic ability of vessels to adjust vascular tone in response to

changes in transmural pressure (Hayreh, 2001).

The retina and ONH circulation exhibit considerable autoregulation

capacity in response to changes in ocular perfusion pressure (Geijer and

Bill, 1979; Riva et al., 1997; Sossi and Anderson, 1983; Pillunat et al.,

1997; Schmidl et al., 2011). Low ocular perfusion pressure - due to

increased IOP and/or low blood pressure - augment the risk of devel-

oping glaucoma as well as disease progression (Bonomi et al., 2000;

Leske et al., 2007, 2008; Quigley et al., 2001; Leske, 2009; Choi et al.,

2007; Sung et al., 2009). Indeed, glaucoma patients display impaired

blood ow autoregulation when the homeostatic limits are challenged

by changes in perfusion pressure, posture, hypercapnia, hyperoxia, or

cold temperatures (Grunwald et al., 1984a; Nagel et al., 2001; Gherghel

et al., 2004; Sines et al., 2007; Feke and Pasquale, 2008; Venkataraman

et al., 2010; Cherecheanu et al., 2012; Prada et al., 2016). For example,

systemic hyperoxia induced by breathing pure oxygen results in marked

vasoconstriction of retinal arterioles and ONH capillaries in healthy

human subjects (Eperon et al., 1975; Riva et al., 1983, 1986b; Stefansson

et al., 1988; Hickam and Frayser, 1966; Jean-Louis et al., 2005; Fallon

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

et al., 1985; Kiss et al., 2002; Shiga et al., 2015). A cross-sectional study

revealed that patients with open-angle glaucoma display weaker vaso-

active responses to systemic hyperoxia than aged-matched controls

(Kiyota et al., 2017). Furthermore, weaker ONH vasoreactivity to sys-

temic hyperoxia was associated with the progression of visual eld de-

fects in patients with open-angle glaucoma (Kiyota et al., 2020).

Dynamic autoregulation refers to the actions causing rapid and

transient vascular changes in response to sudden shifts such as a pressure

step challenge (e.g., a rapid increase of IOP or decrease of blood pres-

sure), reecting the processes that precede the equilibrated steady state

or static autoregulation discussed above (Liang et al., 2010; Prada et al.,

2016). Analysis of dynamic autoregulation involves high temporal res-

olution blood ow measurements, made during the initial phases of

pressure changes, effectively revealing autoregulatory dysfunction

(Noack et al., 2007; van Beek et al., 2008; Liang et al., 2010). Studies of

cerebral blood ow indicate that dynamic autoregulation is a better

indicator of hemodynamic decits than static autoregulation because it

correlates more closely with neuronal activity (Panerai, 1998; Rose-

ngarten et al., 2007). Of interest, elegant studies in non-human primates

subjected to chronic IOP elevation demonstrated ONH blood ow dy-

namic autoregulation dysfunction (Wang et al., 2014b), whereas static

autoregulation remained unchanged (Wang et al., 2014a). Recent work

conducted in a clinical setting with healthy human volunteers also

leveraged the high temporal resolution of the laser speckle owgraphy

(LSFG) imaging technique to evaluate changes in blood ow and pa-

rameters of pulsatility (over the cardiac cycle) within capillaries of the

ONH and the major retinal vessels following acute perturbations of IOP

by ophthalmodynamometry (Iwase et al., 2021). The results demon-

strated that acute IOP elevation caused rapid reduction of blood ow

and higher resistance to blood ow within ONH capillaries, as indicated

by a host of pulsatility parameters (Iwase et al., 2021). Similarly, LSFG

pulsatility parameters, indicative of higher resistance to ow, were

found to be elevated in older age (Luft et al., 2016), an important risk

factor for glaucoma and its progression. LSFG pulsatility was also pre-

dictive of the rate of subsequent progression of visual eld loss in

glaucoma suspects (Gardiner et al., 2023) and was found to be increased

in early stage normotensive glaucoma (Shiga et al., 2013). This pattern

of results parallels altered dynamic autoregulation observed in animal

models (Wang et al., 2014b). It is possible that increased resistance is

caused by mechanically ‘stiffer’ vasculature, loss of capillaries from

normal beds and/or via functional effects such as aberrantly constricted

pericytes (see below). Collectively, these data suggest that blunted ONH

and retinal vasoreactivity contribute to glaucoma pathophysiology and

that dynamic autoregulation is a useful outcome measure to identify

vascular dysfunction in glaucoma patients when conventional ap-

proaches are insufcient.

2.4. Neurovascular dysfunction in glaucoma subjects

Discovered almost 150 years ago, neurovascular coupling (NVC) is a

physiological mechanism that adapts blood ow in accordance with

local neuronal activity, thus bringing additional oxygen and nutrients to

active neurons (Mosso, 1880; Roy and Sherrington, 1890; Friedland and

Iadecola, 1991). This process, also known as functional hyperemia, is

particularly active in the vasculature of the retina and optic nerve in

response to neuronal activation induced by light stimuli (Bill, 1975; Riva

et al., 2005; Srienc et al., 2010; Yu et al., 2019; Wareham and Calkins,

2020). In humans, the NVC response has been studied in retinal and

ONH vessels using ickering light stimulation, which triggers vasodi-

lation and increased blood ow under physiological conditions (Riva

et al., 1986a, 1997, 2005; Polak et al., 2002; Garh¨

ofer et al., 2003,

2005). Another study used adaptive optics to enable visualization of

individual retinal capillaries and quantication of their response to focal

light stimulation in healthy human volunteers, and demonstrated evi-

dence of neurovascular coupling at the level of capillaries, as well as

complex response patterns (inhomogeneity of dilations and

constrictions) suggestive of blood ow redistribution according to local

metabolic demands (Duan et al., 2016). Patients with open-angle glau-

coma present a signicant reduction in icker-evoked dilation of retinal

vessels at the early stages of the disease (Grunwald et al., 1984b;

Garh¨

ofer et al., 2004; Riva et al., 2005; Gugleta et al., 2012). Retinal

vascular responses to icker light also decrease with aging (Gugleta

et al., 2013) and vasospastic propensity (Gugleta et al., 2006), two major

risk factors for glaucoma development (Guedes et al., 2011; Flammer

et al., 2001). Capillaries in the ONH also possess NVC capacity in

response to icker light stimulation (Riva et al., 2005; Gugleta et al.,

2007). Changes in ONH blood ow following luminance icker in

glaucoma patients, including individuals in the initial phases of the

disease are decreased relative to healthy controls (Riva et al., 2004).

Together, these data support the involvement of NVC dysfunction at the

early stages of glaucomatous damage. The next sections discuss the

cellular and molecular mechanisms that regulate NVC and how decits

in these processes can have critical repercussions for glaucoma

pathogenesis.

3. The neurovascular unit: at the heart of stress-related changes

in glaucoma

The function of the retina and optic nerve is not mediated by neurons

alone and, instead, requires multiple cells that communicate with each

other to fulll the metabolic requirement of these tissues through co-

ordinated neurovascular responses. This community of cells is known as

the neurovascular unit (NVU), a concept rst introduced in 2001 by the

Stroke Progress Review Group meeting of the National Institute of

Neurological Disorders and Stroke of the National Institutes of Health to

acknowledge the symbiotic relationship between neurons and sur-

rounding cells (Iadecola, 2017). The NVU in the retina and optic nerve is

composed of neurons, endothelial cells, pericytes, smooth muscle cells,

and glial cells including astrocytes, Müller cells, and microglia (Fig. 2).

The complex signaling between the multiple cell types of the NVU en-

gages distinct effector systems that coordinate neurovascular responses

and maintain the integrity of vascular barriers (Newman, 2013; Iade-

cola, 2017; Sweeney et al., 2019; Pfeiffer et al., 2021; Alarcon-Martinez

et al., 2021; Kugler et al., 2021; O’Leary and Campbell, 2023).

Increased blood ow occurring through the actions of the NVU is also

believed to be important to clear the brain and retina of potentially toxic

by-products of neuronal activity such as lactate and carbon dioxide as

well as for temperature regulation (Zhu et al., 2006; Tarasoff-Conway

et al., 2015). In addition, it is now clear that the NVU interacts bidi-

rectionally with the systemic metabolism, peripheral immune system,

and gut microbiota (Tiedt et al., 2022) expanding its implications in

health and disease. Consistent with this, dysfunction of NVU compo-

nents leading to abnormal vascular responses occurs in several pathol-

ogies including diabetes, Alzheimer’s disease, and other conditions

(Garh¨

ofer et al., 2020; Li et al., 2023). For example, in diabetic reti-

nopathy, pericyte loss and endothelial cell dysfunction coupled with

neuronal and glia alterations are thought to impair functional hyper-

emia (Mishra and Newman, 2010; Garh¨

ofer et al., 2020), highlighting

the involvement of multiple NVU cellular networks in the disease pro-

cess. In addition, pericyte and endothelial cell impairment lead to

capillary constriction during myocardial infarction, stroke, epilepsy, or

COVID-19 infection (Hall et al., 2014; O’Farrell et al., 2017; Leal--

Campanario et al., 2017; Hirunpattarasilp et al., 2023). Endothelial

dysfunction has also been observed in the systemic vasculature, partic-

ularly in patients with low-tension glaucoma (Henry et al., 1999; Su

et al., 2006), suggesting that multi-systemic vascular risk factors may

inuence local ocular NVC breakdown in glaucomatous eyes. Collec-

tively, these studies support the notion that while RGC activity initiates

the process of neurotransmission from retina to brain, the imple-

mentation of a full hemodynamic response requires nely tuned in-

teractions with other NVU cells. In the next sections, we will review the

importance of the primary NVU cellular components in the context of

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

neurovascular regulation and the implications for glaucoma.

3.1. RGCs as orchestra conductors of hemodynamic changes

Neurons are traditionally considered the initiators of the local neu-

rovascular response by signaling to blood vessels, directly or indirectly,

thus triggering coordinated hemodynamic changes (Iadecola, 2017). An

orchestrated neurovascular response is achieved by two means: i)

metabolic negative feedback, and ii) a feedforward mechanism.

Increased energy consumption induced by neuronal activity lowers ox-

ygen and/or glucose leading to the accumulation of metabolic byprod-

ucts, which act as signals for blood vessel dilation required to provide

more energy (metabolism-dependent feedback) (Attwell et al., 2010;

Iadecola, 2017; Pfeiffer et al., 2021). At the same time, glutamate

released by neuronal activity acts on glutamate receptors leading to

calcium (Ca

)-dependent release of vasoactive factors that drive the

initial feedforward mechanism (metabolism independent) of the local

blood ow response (Iadecola, 2017). The metabolism-dependent

feedback and feedforward mechanisms are not mutually exclusive

(Iadecola, 2017), and both are likely to contribute to the vascular energy

supply that supports RGCs depending on the timing, intensity, and

duration of their activation.

The metabolic negative feedback is triggered by a sudden increase in

ATP consumption at the onset of neurotransmission lowering tissue

oxygenation and glucose, while increasing carbon dioxide and meta-

bolic byproducts, thus blood ow is matched with the metabolic need of

active neurons (Iadecola, 2017). In support of this, brain blood ow was

shown to increase in response to a transient decrease in tissue oxygen-

ation (Lecoq et al., 2011; Parpaleix et al., 2013). Studies in the lateral

geniculate nucleus and visual cortex of cats found a reduction in oxygen

and glucose, preceding blood ow increase, at the onset of light-evoked

neural activity (Freeman and Li, 2016). Of interest, analysis of the ce-

rebral microvasculature showed that transient dips in tissue oxygen

tension at the beginning of neuronal activation increased the deform-

ability of red blood cells improving blood ow velocity and capillary

hyperemia (Wei et al., 2016a). In the retina, optic nerve, and brain, the

elevation of the carbon dioxide partial pressure was shown to induce a

global blood ow increase (Harris et al., 1998; Hoiland et al., 2019). In

addition, active neurons release two potent vasodilator byproducts,

adenosine and lactate (Ko et al., 1990; Ido et al., 2001), which can

contribute to NVC by acting as mediators of the metabolic negative

feedback (Attwell et al., 2010; Iadecola, 2017).

Metabolic stress and energy decits have been shown to contribute

to RGC damage in glaucoma (Inman and Harun-Or-Rashid, 2017; Ito and

Di Polo, 2017; Morquette and Di Polo, 2008; Williams et al., 2017a;

Williams et al., 2017b; Belforte et al., 2021, Quintero et al., 2022).

Consistent with this, hyperactivation of the energy sensor adenosine

monophosphate kinase, which is triggered by low intracellular ATP, was

found in RGCs from mice subjected to ocular hypertension and primary

open-angle glaucoma patients (Belforte et al., 2021). While direct proof

of ATP shortage in living RGCs had been lacking, a recent longitudinal

study using two-photon microscopy live imaging demonstrated a sub-

stantial decrease in ATP levels in RGC soma and axons subjected to

ocular hypertensive stress (Quintero et al., 2022). A drop in RGC energy

reserves in glaucoma may compromise the ability of these neurons to

orchestrate NVC regulation restricting the timely delivery of oxygen and

glucose which will, ultimately, lead to neurodegeneration. However, it

is also possible that NVC failure may reduce the energy available to

RGCs (see section 4.4). Recent studies demonstrated that adenosine and

lactate can promote RGC survival (Vohra et al., 2019; Agarwal and

Agarwal, 2021; Boia et al., 2020). Furthermore, it is still unknown

whether endogenous lactate or adenosine, locally produced by RGCs or

surrounding cells, restore NVC responses enhancing neuronal viability

and function during glaucomatous damage. While intracellular ATP

decreases in RGCs subjected to high intraocular pressure (Quintero

et al., 2022), high extracellular ATP has been observed in eyes with

glaucoma (Li et al., 2011; Lu et al., 2015). Excessive extracellular ATP,

released from damaged RGCs and neighboring glial cells, can bind to

P2X7 purinergic receptors on RGCs and microglia (Campagno et al.,

2021; Ishii et al., 2003; Puthussery and Fletcher, 2004) triggering

neuronal death (Hu et al., 2010). Given that excess extracellular ATP is

involved in pericyte-dependent capillary contraction via purinergic

signaling activation in brain diseases such as Alzheimer’s disease

(Hørlyck et al., 2021), abnormal extracellular ATP levels associated with

glaucomatous stress may harm microvasculature as well. Additional

studies are needed to fully understand the link between RGC metabolic

stress and NVC impairment in glaucoma.

In the brain, feedforward regulation is primarily driven by the

release of potent vasodilators, notably nitric oxide (NO) and prosta-

glandins (Yang et al., 2003; Attwell et al., 2010; Lecrux and Hamel,

2016). Neuronal activity-evoked ionic changes such as an increase in K

can also mediate NVC (Longden et al., 2016) and will be discussed in the

context of the endothelial cell responses in the next section. Early studies

showed that NO increased following light icker stimulation in the

retina and ONH (Donati et al., 1995; Buerk and Riva, 2002) lending

support to a role for NO in neurovascular changes at these locations.

Consistent with these ndings, studies in cats as well as humans showed

that systemic administration of non-selective NOS inhibitors reduced

icker-evoked increases in blood ow (Kondo et al., 1997; Dorner et al.,

2003). More recent studies suggest that NO does not mediate NVC in the

retina (Metea and Newman, 2006a; Nippert and Newman, 2020) but

rather serves as a vasomodulator, similar to its role in the cerebral cortex

(Lindauer et al., 1999). For example, when NO levels were increased or

decreased in the medium of acutely isolated whole-mounted retinas, it

Fig. 2. Cellular components of the neurovascular unit (NVU) in the retina and optic nerve. (A) Schematic representation of the cells that form the neurovascular unit

in the retina, which include retinal ganglion cells (RGC), astrocytes, endothelial cells, pericytes, Müller cells, and microglia. (B) The neurovascular unit in the optic

nerve includes RGC axons, both the unmyelinated and myelinated portions, astrocytes, pericytes, endothelial cells, microglia, and oligodendrocytes, the latter

depending on whether the axon segment is myelinated or not. Created with BioRender.com.

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

promoted light-evoked arteriole vasoconstriction or vasodilation,

respectively (Metea and Newman, 2006a), possibly by modulating the

synthesis of arachidonic acid derivatives (Attwell et al., 2010). NO can

stimulate the production of cyclic guanosine monophosphate (cGMP),

which inhibits Ca

entry in pericytes hence reducing their contractility

(Sakagami et al., 2001). Thus, NO can function as a mediator of NVC and

has been implicated in glaucomatous neurodegeneration (Haider et al.,

2022).

In models of diabetic retinopathy, loss of NVC has been attributed to

an increase in NO, which blocked the production of vasodilation agents

derived from Müller cells (see Section 3.4.1.). In support of this,

inducible NO synthase (NOS) inhibitors restored functional hyperemia

in retinal explants (Mishra and Newman, 2010; Mishra et al., 2011). The

role of NOS in glaucomatous optic neuropathy has been controversial.

Early studies showed increased expression of inducible NOS in the ONH

of open-angle glaucoma patients (Neufeld et al., 1997), and NOS inhi-

bition with aminoguanidine provided RGC neuroprotection in a rat

glaucoma model (Neufeld et al., 1999). However, subsequent studies

using several independent quantitative methods and robust glaucoma

models failed to identify signicant changes in NOS expression in the

retina, ONH, and optic nerve; nor was neuroprotection conferred by

genetic deletion or pharmacological inhibition of NOS (Pang et al.,

2005; Libby et al., 2007). These ndings, together with studies showing

that NO inhibition only partly accounts for NVC (Hosford and Gourine,

2019), suggest that other cellular and molecular pathways of neuro-

vascular regulation are at play and will be discussed in the following

sections.

3.2. Endothelial cells set the tone

The wall of blood vessels is lined by a thin monolayer of endothelial

cells, known as the vascular endothelium, which form an adaptive and

dynamic interface between the blood circulation and the tissue envi-

ronment (Krüger-Genge et al., 2019). Endothelial cells are subjected to

hemodynamic changes and receive and send signals to regulate the

myogenic or vessel tone, vascular permeability, and immune responses

(Loh et al., 2018; O’Leary and Campbell, 2023). Cells in the vascular

endothelium are electrically coupled through gap junctions, which

allow the transfer of electrical signals from capillaries to arterioles and

venules (Zhang et al., 2011; Longden et al., 2017). Being in close contact

with mural cells, namely smooth muscle cells and pericytes, endothelial

cells are active players within the NVU (Dalkara and Alarcon-Martinez,

2015). The myogenic response is determined by the balance between

vasodilation and vasoconstriction signals that act on receptors expressed

by endothelial and mural cells (Davis and Hill, 1999; Koide et al., 2018).

Vasodilation is mediated by endothelium-derived relaxing factors

including endothelial NO (Zhao et al., 2015a; Loh et al., 2018), pros-

tacyclin (Ingerman-Wojenski et al., 1981, Oudemans-Van Straaten et al.,

2010), endothelium-derived hyperpolarizing factor (F´

el´

etou and Van-

houtte, 2006), and hydrogen sulde (Zhao and Wang, 2002), while

vasoconstriction is regulated by other mediators such as angiotensin II,

generated from the endothelium-located angiotensin-converting

enzyme that converts angiotensin I into angiotensin II (Benigni et al.,

2010; Vanlandewijck et al., 2018), thromboxane A

(Ramadan et al.,

1990; Ingerman-Wojenski et al., 1981), endothelin (Vanhoutte and

Tang, 2008; Krüger-Genge et al., 2019), prostaglandin H

(Davidge

et al., 1995), and reactive oxygen species (ROS) (Fan et al., 2014).

Among these, endothelin deserves special attention based on accumu-

lating evidence suggesting a role in glaucoma (Chauhan, 2008; Prasanna

et al., 2011).

Originally isolated from the supernatant of cultured porcine endo-

thelial cells, endothelin-1 (ET-1) is a potent vasoconstrictor with long

lasting effects (Yanagisawa et al., 1988). Two additional family mem-

bers, endothelin-2 (ET-2) and endothelin-3 (ET-3), were identied in

humans (Inoue et al., 1989). Endothelins bind to and activate the

G-protein-coupled receptors ET

and ET

(Arai et al., 1990; Sakurai

et al., 1990) with ET

showing higher afnity for ET-1 and ET-2 than

ET-3 (Arai et al., 1990). ET-1 is presumed to promote vasoconstriction

by acting on ET

receptors expressed in mural cells, notably pericytes

(Howell et al., 2014; Vanlandewijck et al., 2018). Endothelins and their

receptors are also expressed by other cells in the retina and optic nerve,

including RGCs and glia (MacCumber and D’Anna, 1994; Rattner and

Nathans, 2005; Minton et al., 2012; Rattner et al., 2013; Howell et al.,

2014; Patel et al., 2014). ET-1 levels increase in the aqueous humor of

primary open-angle and exfoliation glaucoma patients (Tezel et al.,

1997; Koukoula et al., 2018). Intravitreal injection or overexpression of

endothelins promote RGC death, whereas inhibition of endothelin

signaling is neuroprotective (Orgül et al., 1996; Chauhan et al., 2004;

Sasaoka et al., 2006; Lau et al., 2006; Howell et al., 2011, 2012; Mi et al.,

2012; Blanco et al., 2017; Marola et al., 2020, 2022). However, the

precise site of action of endothelins and how they promote RGC death is

still poorly understood. Of interest, a recent study demonstrated that

cell-specic conditional deletion of both ET

and ET

from RGCs and

macroglia did not prevent ET-1-induced RGC loss (Marola et al., 2022).

In contrast, deletion of ET

from vascular mural cells was sufcient to

block vasoconstriction and RGC death caused by ET-1 exposure (Marola

et al., 2022). Therefore, ET-1, independently of where it is produced,

does not act directly on RGCs or glia but rather uses vascular mural cells

to drive RGC death. These ndings suggest that ET

-mediated capillary

vasoconstriction is a critical event leading to RGC loss.

Although endothelial cells have been traditionally considered to play

a limited role in NVC, recent elegant studies have challenged this idea by

reporting that endothelial cells express receptors and channels that

transduce neuronal activity into a vascular response (Longden et al.,

2016, 2017, 2021; Harraz et al., 2018a,b; Zhang et al., 2021; Thakore

et al., 2021; Sancho et al., 2022b). In brain vessels, potassium (K

) has

been proposed to be an important mediator of NVC acting on the strong

inward-rectier K

) channels in the vascular wall, which serve as

sensors of external K

(Longden and Nelson, 2015). Neuronal activity

increases extracellular K

and activation of K

channels, predominantly

the K

2.1 subtype expressed in both endothelial and mural cells

(Bradley et al., 1999; He et al., 2018), leading to hyperpolarization and

pronounced vasodilation (Longden and Nelson, 2015; Longden et al.,

2016). This capillary response propagates retrogradely across endothe-

lial cells through gap junctions, dilating upstream arterioles and

increasing blood ow along the entire vascular tree (Longden et al.,

2017; Moshkforoush et al., 2020; Zhang et al., 2021). K

2.1 dysfunction

has been associated with a number of pathologies including chronic

stress, cerebral small vessel disease, and traumatic brain injury (Long-

den et al., 2014; Dabertrand et al., 2021; Weir and Longden, 2021). Low

levels of phosphatidylinositol 4,5-bisphosphate (PIP

), an intracellular

phospholipid that is essential for K

2.1 channel function, have been

reported in a model of small vessel disease (Harraz et al., 2018a,b), and

PIP

administration was sufcient to restore K

2.1 channel signaling

and cerebral blood ow in response to neuronal activation (Dabertrand

et al., 2021). K

2.1 channels are expressed in the retina and optic nerve

(Brasko and Butt, 2018; Klose et al., 2021), however, studies on the role

of Kir2.1 signaling in endothelial cell hyperpolarization in the context of

glaucoma and their implication in NVC dysfunction are still lacking.

3.3. Pericytes: location, location, location

Described over 150 years ago, pericytes are cells that wrap tightly

around the surface of capillaries in a bump-on-a-log fashion (Eberth,

1871; Rouget, 1873; Attwell et al., 2016). Pericytes are a heterogeneous

population of mesenchymal cells that can have different morphologies

and functions depending on their location (Bohannon et al., 2021;

Garrison et al., 2023). In the brain, two main structural and functional

pericyte types have been described downstream of the penetrating

arteriole: ensheathing pericytes on the arteriole-capillary transition, and

thin-strand capillary pericytes located on capillaries (Hartmann et al.,

2022). Pericytes at the arteriole-capillary transition, also known as

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

ensheathing pericytes, display a mesh-like pattern of short-range pro-

cesses wrapped around the vessels (Grant et al., 2019; Alarcon-Martinez

et al., 2021; Longden et al., 2023). In contrast, capillary pericytes extend

thin processes along the capillary surface that can reach hundreds of

microns in length (Berthiaume et al., 2018; Alarcon-Martinez et al.,

2021). The human retinal vasculature is composed primarily of capil-

laries and ~95% of those are wrapped by pericytes (Chan-Ling et al.,

2011; Trost et al., 2019). Therefore, here we will use the term ‘pericyte’

to refer to capillary pericytes, which will be the focus of this and sub-

sequent sections (see sections 4 and 5.2).

Capillary pericytes are embedded within the basement membrane of

the capillary wall with their soma and processes in intimate contact with

adjacent endothelial cells (Mandarino et al., 1993). Indeed, pericyte thin

processes are connected with more than one endothelial cell via gap

junctions as well as peg-and-socket interdigitations, where pericytes or

endothelial cells send a projection to make direct contact with the

adjacent cell (Ornelas et al., 2021). These contact points are likely to be

sites of gap junction communication and cross-talk between pericytes

and endothelial cells, where key signaling events take place including

increases in local Ca

and second messengers (Cuevas et al., 1984;

Armulik et al., 2005). In whole retinas, functional connectivity maps

show that pericytes establish gap junction connections primarily with

neighboring pericytes and endothelial cells, and much less with arteri-

olar smooth muscle cells (Kovacs-Oller et al., 2020). Moreover, pericytes

can interact with surrounding neurons and glia (Allsopp and Gamble,

1979; Hogan and Feeney, 1963; Maynard et al., 1957; Murakami et al.,

1979; Sims, 1986; Ushiwata and Ushiki, 1990). For example, in human

retinas, tight contacts between glial cells and pericytes have been re-

ported in addition to fenestrations in the basement membrane of peri-

cytes at the contact site with Müller glia (Hogan and Feeney, 1963). The

central location of pericytes, which readily allows them to respond to

vasodynamic factors derived from neurons and other cells in the NVU,

makes them ideal candidates to regulate capillary blood ow and NVC

(Fig. 2). Consistent with this, single-cell transcriptomic studies demon-

strate that capillary pericytes express multiple ion channels and G

protein-coupled receptors for vasoactive mediators (Hariharan et al.,

2020; Vanlandewijck et al., 2018). Therefore, pericytes are ideally

positioned to play a critical role in regulating blood ow to meet the

energy requirements of RGCs.

Until recently, and despite their strategic location and vasodynamic

responses, pericytes were not considered serious contenders to regulate

blood ow in vivo, a process that was attributed solely to arteriolar

smooth muscle cells (Hill et al., 2015; Pfeiffer et al., 2021; Hartmann

et al., 2022). The controversy to resolve this issue stemmed from mul-

tiple challenges including a lack of tools to unequivocally identify and

target capillary pericytes, low spatial resolution to visualize small

changes in capillaries, and failure to detect components of the con-

tractile machinery required to constrict microvessels. However, recent

advances in the eld have yielded new data strongly supporting that

pericytes are contractile and that they play key roles in the regulation of

microcirculatory blood ow in the brain and retina (Peppiatt et al.,

2006; Hall et al., 2014; Kisler et al., 2017; Alarcon-Martinez et al., 2018,

2019, 2020, 2022; Vanlandewijck et al., 2018; Nortley et al., 2019;

Gonzales et al., 2020; Hartmann et al., 2021; Longden et al., 2023). For

example, histological and single-cell transcriptomic studies show that

pericytes express genes involved in actomyosin contraction, such as

lamentous actin (F-actin), myosin, regulators of myosin phosphoryla-

tion, and L-type voltage gated Ca

channels (Vanlandewijck et al.,

2018; Kureli et al., 2020). However, expression of smooth muscle actin

organized in alpha conformation (

-SMA), required to bind myosin

proteins, was reported to be heterogeneous among pericyte populations

and was often found to be present at low or undetectable levels (Nehls

and Drenckhahn, 1991; Bandopadhyay et al., 2001). A recent study in

the retina addressed this issue by showing that when F-actin depoly-

merization is prevented using stabilizing agents or by snap xation,

-SMA is reliably detected in a large population of pericytes in all retinal

plexuses (Alarcon-Martinez et al., 2018). Furthermore, when

-SMA

expression was blocked, the ability of retinal pericytes to constrict

capillaries was markedly reduced (Alarcon-Martinez et al., 2018, 2019).

Recent functional studies have unequivocally established a critical role

for pericytes in the regulation of blood ow and NVC in the retina and

optic nerve, both in physiological and pathological conditions, including

glaucomatous damage (Alarcon-Martinez et al., 2020, 2022) and will be

discussed in Section 4.

3.4. Glia-derived vasoactive molecules

Once believed to play only supportive roles for neurons, glial cells

are now recognized to have a major inuence on most physiological

aspects of brain function including neurotransmission, energy homeo-

stasis, and blood ow (Kugler et al., 2021; Nampoothiri et al., 2022).

Glial cells in the mammalian retina and optic nerve, comprising astro-

cytes, Müller cells, microglia, and oligodendrocytes, play multiple roles

in the regulation of visual function (Yazdankhah et al., 2021; Benfey

et al., 2022). These cells are equipped with the molecular machinery to

sense and decode neuronal activity (Benfey et al., 2022). By responding

to multiple neurotransmitters, glial cells undergo coordinated changes

in intracellular Ca

that promote the release of potent glia-derived

vasoactive agents (Newman, 2015). The physical proximity of glial

cells with pericytes and capillaries as well as their ability to respond to

neurons, make them ideal hubs for the tight control of blood ow

required to support optimal neural circuit function.

3.4.1. Signals from Müller cells and astrocytes

Müller cells are among the rst responders following retina or optic

nerve injury and can have neuroprotective as well as detrimental effects

on RGCs depending on the stress stimuli and timing of injury (Newman

and Reichenbach, 1996; Di Polo et al., 1998; Gauthier et al., 2005; Joly

et al., 2007; Pernet et al., 2007; Lebrun-Julien et al., 2009a,b, 2010;

Cueva Vargas et al., 2015, 2016). Most of our existing knowledge on

how glia regulate NVC in the visual system stems from studies focused

on the role of Müller glia (Newman, 2015; Biesecker et al., 2016; Nippert

and Newman, 2020), whereas not much is currently known about the

role of astrocytes in this response. Müller cells are ideally positioned to

act as intermediaries in NVC because their ne processes surround

retinal neurons and synapses as well as capillaries in all plexuses, which

can readily respond to vasoactive agents. Indeed, stimulation of Müller

cells in retinal explants evoked arteriole dilation demonstrating that

these glial cells release vasodilating factors (Metea and Newman, 2006a;

Mishra and Newman, 2010). Subsequent experiments showed that

light-evoked vasodilation is abolished when neuron-to-Müller glia

signaling is impaired. For example, when ATP receptors on Müller cells

were blocked using purinergic antagonists, light-evoked Ca

increase

in Müller cells was eliminated thus preventing vessel dilation despite

persistent neuronal activity (Metea and Newman, 2006a). Light stimu-

lation increased Ca

in Müller cell endfeet in contact with capillaries,

and capillary dilation was abolished when glial Ca

signaling was

reduced (Biesecker et al., 2016). Although earlier studies reported a

correlation between increased Ca

in Müller cells and vasodilation, the

current consensus is that Ca

rises in Müller cells and brain astrocytes

exert dual effects - vasodilation or vasoconstriction - depending on

several parameters including pre-existing vessel tone, NO and O

levels

as well as the type of vasomodulators produced (Mulligan and MacVicar,

2004; Metea and Newman, 2006b; Attwell et al., 2010; Rungta et al.,

2016; Blanco et al., 2008; Chuquet et al., 2007).

Studies using isolated retina and brain slice preparations support a

model in which active neurons, with concomitant glutamate and/or ATP

release, signal Müller cells or astrocytes to increase Ca

and produce

vasomodulators, which then act on vessels to regulate blood ow (Att-

well et al., 2010). Indeed, increased neuronal function boosts Ca

levels

in Müller glia promoting the activation of phospholipase A

and syn-

thesis of arachidonic acid metabolites, which are potent vasomodulators

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Progress in Retinal and Eye Research 97 (2023) 101217

(Newman, 2015). The accumulation of arachidonic acid leads to the

production of metabolites such as prostaglandins (e.g. PGE

), epox-

yeicosatrienoic acids (EETs), and 20-hydroxy-eicosatetraenoic acid

(20-HETE), which can dilate or constrict nearby vessels (Zonta et al.,

2003; Metea and Newman, 2006b; Gordon et al., 2008). PGE

promotes

vasodilation by binding to the EP4 prostaglandin receptor (Yokoyama

et al., 2013), which stimulates adenylyl cyclase and increases cAMP

production to decrease intracellular Ca

levels in mural cells (e.g.

smooth muscle cells and pericytes) (Mori et al., 2007; Misfeldt et al.,

2013). Conversely, PGE

can also induce vasoconstriction through

activation of the receptor for thromboxane, a vasoconstricting deriva-

tive of arachidonic acid, or the E prostanoid receptor-3 presumably by

decreasing cAMP levels (Abran et al., 1995; Wright and Harris, 2008;

Torring et al., 2014). EETs contribute to vasodilation by activating

-dependent K

channels on vascular smooth muscle and endothelial

cells resulting in membrane hyperpolarization and relaxation (Campbell

and Fleming, 2010; Mori et al., 2021). Regarding the arachidonic acid

metabolite 20-HETE, this molecule was shown to promote light-evoked

vasoconstriction in isolated whole-mount retinas (Metea and Newman,

2006b). This nding is consistent with studies using hippocampal slices

showing that glial stimulation resulted in vasoconstriction mediated by

20-HETE (Mulligan and MacVicar, 2004).

In summary, glial cells in the retina respond rapidly to neuronal

activity by producing vasoactive agents including: i) prostaglandins,

which can exert either vasodilating or vasoconstricting responses, ii)

EETs which are vasodilating, and iii) 20-HETE which is vasoconstricting.

The limitations of these earlier studies is that they utilized whole-mount

preparations ex vivo, therefore little or no information from in vivo

studies exists. In addition, most of the studies examined the response of

retinal arterioles to glia-derived vasoactive factors. However, the

response of capillaries, which represent the vast majority of the retinal

microvasculature, remains poorly understood. Furthermore, there is

currently a lack of information about the role of Müller cell- or astrocyte-

derived vasomodulators in the context of glaucomatous damage,

through known or yet unknown factors, presenting a major gap in our

understanding of how glia contribute to NVC dysregulation in this

disease.

3.4.2. The role of microglia

Microglia are highly specialized resident macrophages ideally posi-

tioned to survey the central nervous system environment due to their

highly dynamic processes, plasticity, longevity, motility, and ability to

respond to many stimuli (Prinz et al., 2021). Microglia play critical roles

in the maintenance of tissue homeostasis during development, health,

and disease including neurogenesis, myelination, vasculogenesis, and

circuit renement (Hammond et al., 2018). The traditional view of

‘good’ versus ‘bad’ microglia has been revised to include the multiple

variables that drive phenotypic transformation of microglia in myriad

conditions and at multiple levels including transcriptional, epigenetic,

translational, and metabolic (Paolicelli et al., 2022). Genetic depletion

of microglia expressing the C-X3-C motif chemokine receptor (CX3CR1)

did not alter the vascular pattern and laminar organization of the retina,

and retinal neurons did not undergo overt death (Wang et al., 2016).

However, the absence of microglia negatively affected synaptic trans-

mission and light-evoked retinal function (Wang et al., 2016). A recent

study showed that microglia depletion using PLX5622, a pharmacolog-

ical inhibitor of the colony-stimulating factor 1 receptor (CSF1R) that is

essential for microglia survival, exacerbated RGC loss during ocular

hypertension without compromising the pattern electroretinogram

(ERG) amplitude or visual acuity (Tan et al., 2022) supporting the

functional diversity of microglia. Consistently, studies on the role of

microglia in models of optic nerve injury including glaucoma have been

reported to be deleterious or benecial depending on the context. This

subject has been covered in recent reviews and will not be discussed here

(Ahmad and Subramani, 2022; Pitts and Margeta, 2023). Instead, we

will focus on the emerging role of microglia in the regulation of vascular

responses.

The role of microglia on NVC regulation and the mechanisms

involved are still poorly understood, but recent work suggests that these

cells interact with other members of the NVU to modulate blood ow.

Microglial processes are in dynamic contact with every cell type of the

NVU (Csaszar et al., 2022), making microglia ideal components to in-

uence and regulate vascular function. During development, microglia

play a critical role in vessel formation and modulate the sprouting,

migration, anastomosis and renement of the retinal vasculature

(Arnold and Betsholtz, 2013). Studies in which microglia were depleted

demonstrated that they play a key role in angiogenesis and the forma-

tion of new vessels (Checchin et al., 2006; Li et al., 2019) by secreting

soluble factors such as the Fas ligand (CD95L), which binds to its re-

ceptor on the endothelial cell’s lopodia (Chen et al., 2017). Further,

activation of the Notch signaling pathway by the MAS1 oncogene (MAS

receptor), the receptor of angiotensin-(1–7), has been shown to be

necessary for the recruitment of microglia around vessels to promotes

vascular growth in the developing retina (Foulquier et al., 2019; Outtz

et al., 2011). Microglia establish connections with pericytes and endo-

thelial cells early during development and, when pericytes are depleted,

the density and proliferation of microglia is markedly reduced (Hattori

et al., 2022) suggesting a critical crosstalk between microglia and

pericytes.

Microglia have been proposed to modulate vascular responses in a

context-dependent manner. For example, activation of the microglial

fractalkine-CX3CR1 signaling leads to capillary constriction through the

renin-angiotensin system, a pathway implicated in diabetic retinopathy

(Mills et al., 2021). Microglia depletion with a CSF1R inhibitor

(PLX3397) increased capillary diameter and cerebral blood ow (Bisht

et al., 2021). In contrast, a recent study reported that microglia ablation

impaired functional hyperemia by reducing cerebral blood ow

following whisker stimulation (Cs´

asz´

ar et al., 2022). In pathological

conditions, pathogen-activated microglia secrete pro-inammatory cy-

tokines that alter endothelial cell function (Rodríguez et al., 2022),

which is likely to disrupt neurovascular coupling. Some

microglia-vascular interactions are thought to be mediated by puriner-

gic signaling (Bisht et al., 2021; Cs´

asz´

ar et al., 2022), nonetheless, the

underlying mechanisms are far from being understood. The G-protein

coupled P2Y12 purinergic receptor (P2RY12) is expressed by microglia

(Zhang et al., 2014; Lou et al., 2016) and regulates microglial-directed

motility and migration in response to cell injury (Haynes et al., 2006).

Recent studies using conditional knockout mice for P2RY12 and the

purine channel pannexin 1 (PANX1), the main ATP release channel,

suggest that a microglia-dependent mechanism controls cerebral blood

ow (Bisht et al., 2021; Cs´

asz´

ar et al., 2022). Experiments in mice that

selectively targeted microglia by chemogenetics led to the disruption of

hemodynamic processes in response to ATP (Cs´

asz´

ar et al., 2022).

Moreover, in a model of cerebrovascular adaptation generated by

common carotid artery occlusion, microglia purinergic signaling was

necessary for normalization of cerebral blood ow, and microglia

depletion or blockade of P2RY12 led to decits in adaptation after

repeated vessel occlusion (Cs´

asz´

ar et al., 2022). Collectively, these

studies suggest that microglia may regulate NVC through purinergic

signaling. However, since purine and purinergic receptors are also

expressed by different cell types in the NVU, such as pericytes, endo-

thelial cells, and astrocytes (Hørlyck et al., 2021), further studies are

needed to unravel the precise mechanisms underlying the role of puri-

nergic signaling in blood ow regulation. Studies that directly assess the

role of microglia on NVC dysfunction in experimental glaucoma are

currently lacking.

4. Pericytes are key regulators of neurovascular coupling

4.1. How do pericytes regulate capillary blood ow and NVC?

Accumulating evidence using state-of-the-art approaches to

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Progress in Retinal and Eye Research 97 (2023) 101217

selectively manipulate pericytes in vivo demonstrate that these cells play

a critical role in the regulation of microcirculatory blood ow in both

retina and brain (Alarcon-Martinez et al., 2020, 2022; Hartmann et al.,

2022; Longden et al., 2023). Studies using optogenetics to selectively

stimulate brain pericytes showed that when these cells contract there is a

reduction in blood cell velocity and ux (Nelson et al., 2020; Hartmann

et al., 2021). In contrast, studies using optical ablation of cortical peri-

cytes led to the dilation of capillaries lacking pericyte coverage (Ber-

thiaume et al., 2018), a response that was also observed following

pericyte loss in a model of epileptic seizures (Arango-Lievano et al.,

2018). In the retina, laser-mediated ablation of specialized processes

connecting pericytes blocked NVC coupling (Alarcon-Martinez et al.,

2020), and pericyte contraction reduced capillary blood ow and NVC

regulation during glaucomatous damage (Alarcon-Martinez et al.,

2022). In a complementary study, optogenetic hyperpolarization of

retinal pericytes by selective expression of halorhodopsin, enhanced

capillary blood ow in vivo (Ivanova et al., 2021). Collectively, these

studies strongly support the view that pericytes play critical roles in the

hemodynamic regulation of capillary blood ow in physiological and

pathological conditions.

The nexus between neuronal activation and capillary responses has

been proposed to occur via receptors and channels located on pericytes.

Recent single cell-RNAseq analysis revealed that pericytes express

metabotropic, purinergic, prostanoid, chemokine, adenosine, and

adrenergic receptors; as well as K

, transient receptor potential, Ca

and chloride channels (Vanlandewijck et al., 2018; Hariharan et al.,

2020; Yang et al., 2022). Neuronal- and glia-derived products can

activate these receptors/channels to initiate intracellular pathways

triggering membrane changes and Ca

-dependent

-SMA variations to

promote pericyte contraction or relaxation, hence modulating capillary

diameter and blood ow (Puro, 2007; Peppiatt et al., 2006; Hamilton

et al., 2010; Hariharan et al., 2020). Based on this, the regulation of Ca

levels within pericytes is essential to fulll their role as active regulators

of capillary hemodynamics. The primary pathways of Ca

increase in

pericytes are voltage-dependent Ca

channels (VDCC), the primary

gates of extracellular Ca

into pericytes; and inositol 1,4,5-trisphos-

phate receptors (IP3Rs), which extrude Ca

from the endoplasmic re-

ticulum to the cytosolic space (Burdyga and Borysova, 2018). VDCCs are

regulated by membrane potential changes and are typically composed of

distinct subunits (i.e.,

, β,

δ, and γ). Capillary pericytes are known to

express genes encoding the

subunits for L-type (Ca

1.2, Ca

1.3),

P/Q-type (Ca

2.1), and T-type (Ca

3.1, Ca

3.2) channels as well as genes

encoding the β and

δ auxiliary subunits (He et al., 2018; Vanlande-

wijck et al., 2018). In the retina, L-type VDCCs containing the Ca

1.2

subunit play critical roles in Ca

signals that regulate

pericyte-mediated capillary constriction in experimental glaucoma

(Alarcon-Martinez et al., 2022). In addition, pericytes express transient

potential (TRP) channels (Vanlandewijck et al., 2018), a family of

non-selective ion channels located on the plasma membrane of many

cells that conduct signicant amounts of Ca

(Per´

alvarez-Marín et al.,

2013; Ryskamp et al., 2011; Kriˇ

zaj et al., 2023). Among TRPs, the

vanilloid-type members TRPV1 and TRPV4 have been associated with

glaucomatous damage (Kriˇ

zaj et al., 2023). Indeed, genetic deletion of

TRPV1 reduced RGC somatic size, dendritic length as well as

complexity, and reduced spontaneous activity selectively in OFF

RGCs

(Risner et al., 2020) suggesting the involvement of TRPV1-mediated

pathways on RGC function. However, the role of TRP channels on per-

icyte function and their impact during glaucomatous damage is

currently unknown.

The predominant ion channel expressed by brain pericytes is the

ATP-sensitive K

ATP

) channel, containing the inward rectier (K

)

subunit K

6.1, which accounts for nearly half of all ion channels

expressed by these cells (He et al., 2018; Vanlandewijck et al., 2018;

Hariharan et al., 2020; Sancho et al., 2022a; Ando et al., 2022). A recent

elegant study demonstrated that capillary pericytes act as metabolic

sentinels through K

ATP

channels, which respond to changing energy

levels, to regulate blood ow (Hariharan et al., 2022). Indeed, when

glucose is abundant and ATP levels are high in pericytes, K

ATP

channels

remain closed, but when glucose is low and ATP levels drop, K

ATP

channels open causing K

outow and pericyte membrane hyperpolar-

ization (Hariharan et al., 2022). The hyperpolarizing electrical signal

from pericytes is fed into the underlying endothelium, where it activates

endothelial cell K

2.1 channels promoting upstream transmission to

dilate the penetrating arteriole and increase blood ow to replenish

glucose delivery (Hariharan et al., 2022).

The functional role of vasomodulators on the battery of receptors and

channels expressed by retinal pericytes remains poorly understood,

however, correlative information obtained by application of agonists

and antagonists has shed information on the response of pericytes. For

example, application of ATP or purinergic receptor agonists on isolated

whole retinas induced the contraction of pericytes soma around capil-

laries (Kawamura et al., 2002; Peppiatt et al., 2006) presumably through

a decrease in cAMP production via G protein signaling. Whether

ATP-induced pericyte contraction occurs via a direct or indirect mech-

anism is currently unknown. Arachidonic acid derivatives produced by

glial cells such as the prostaglandin PGE

can be further processed into

thromboxane A

, which can act on thromboxane receptors on pericytes

to promote contraction. In the retina, application of a synthetic analog of

PGE

led to marked pericyte contraction, which was blocked with in-

hibitors of actin depolymerization such as cytochalasin D and latrun-

culin B (Gonzales et al., 2020). Capillary pericytes also express high

levels of the ET-1 receptor A (ednra) (Vanlandewijck et al., 2018), and

display robust contraction in response to ET-1 (Zambach et al., 2021).

Although the mechanism by which ET-1 receptor signaling causes per-

icyte contraction has not been fully elucidated, it is presumed to be the

result of Ca

increase in pericytes through opening of membrane

channels and/or intracellular stores leading to activation of their con-

tractile machinery. Studies in the retina suggest that ET-1 leads to per-

icyte contraction via multiple pathways including inhibition of

ATP-sensitive K

channels, which prevent hyperpolarization and peri-

cyte dilation, and reduced gap junction connectivity between pericytes

(Kawamura et al., 2002). In terms of pericyte relaxation leading to

vasodilation, several pathways have been identied including glutamate

(Hall et al., 2014) and adenosine, the latter likely promoting pericyte

relaxation by acting on A2a purine receptors (adora2) leading to

ATP-dependent K

channel ux (Li and Puro, 2001). These responses

require a high level of communication between pericytes and other cells

in the NVC unit as well as cross-talk between pericytes, which will be

described in the next section.

4.2. Discovery of inter-pericyte tunneling nanotubes (IP-TNTs)

The previous section covered a wealth of information supporting the

concept that pericytes regulate microcirculatory blood ow by con-

tracting and relaxing to induce changes in capillary diameter. However,

this model does not account for the need to coordinate dilation and

constriction across capillary networks to achieve the ne distribution of

blood supply required by active RGCs. This is particularly important in

the retina where patterns of visual stimulation imply that some neurons

are more active than others at any given time (Westheimer, 2007), thus

blood must be rapidly relocated to meet the demand of light-activated

versus light-inactivated neurons. Until recently, the mechanisms un-

derlying retinal blood distribution were poorly understood. Recent ad-

vances in this regard include the discovery of tunneling nanotube

(TNT)-like processes, which connect two bona de pericytes to form a

functional hemodynamic network in the mouse retina (Alarcon-Marti-

nez et al., 2020). These highly specialized microstructures, termed

inter-pericyte tunneling nanotubes (IP-TNTs), are extremely ne pro-

cesses that connect two distinct pericytes located on separate capillary

systems (Fig. 3). IP-TNTs emerge from the soma of a pericyte, named

proximal pericyte, and extend to a neighboring capillary where they

establish connections with a distal pericyte. They are abundantly

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Progress in Retinal and Eye Research 97 (2023) 101217

expressed in the mouse retina and optic nerve head as well as visual

centers in the brain (Fig. 4). Three-dimensional reconstruction revealed

that IP-TNTs are tubular, with an average diameter of ~500 nm that can

extend between 4

m and 90

m in length, and are abundant in all

retinal plexuses (Alarcon-Martinez et al., 2020). TNTs have been

described in other systems, including tumor and immune cells, and a

conserved structural requirement is that they contain F-actin (Yamashita

et al., 2018; Tarasiuk and Scuteri, 2022). Consistently, IP-TNTs are

endowed with an F-actin cytoskeleton as well as

-SMA, focal adhesion

kinase 1, and ribosomal protein S6, but they lack

-tubulin and

mesenchymal stem cell markers (Alarcon-Martinez et al., 2020).

IP-TNT-specic markers have yet to be identied.

TNTs have been proposed as a novel means for cell-cell communi-

cation and potential transfer of biological cargos between distant cells,

thus a phenotypic requirement is that they must connect two cells

(Mittal et al., 2019; Ortin-Martinez et al., 2021). Experiments using

single-pericyte electroporation demonstrated that low molecular weight

uorescent dyes (e.g. uorescein, 332 Da) diffused rapidly from the

proximal pericyte soma through the IP-TNT and into the soma of the

distal pericyte (Alarcon-Martinez et al., 2020) (Fig. 5A). In contrast,

larger molecular weight markers (e.g., dextran, 3 kDa) diffused within

the proximal pericyte, but did not enter the distal pericyte suggesting the

existence of gap junctions at this interface. When a low molecular

weight marker was electroporated into the proximal pericyte in the

presence of the gap junction blocker carbenoxolone (CBX), it accumu-

lated in the soma and IP-TNT (Fig. 5B). Dextran accumulation in the

proximal pericyte allowed unmistakable identication of two

IP-TNT-coupled pericytes as well as their connection at the level of the

IP-TNT terminal (end-foot) and the distal pericyte process

(Alarcon-Martinez et al., 2020). Ultrastructural analysis using focused

ion beam electron microscopy (FIB-SEM) further conrmed that IP-TNT

end-feet connect with distal pericyte processes through

membrane-to-membrane contacts evocative of gap junctions (Fig. 5C)

(Alarcon-Martinez et al., 2020). Consistent with this, expression of

connexin 43 (Cx43) was detected at IP-TNT end-feet (Alarcon-Martinez

et al., 2020) (Fig. 5D). Together, these data show that IP-TNTs are

close-ended processes in which the cytoplasm of the two linked pericytes

remain separate, but ions and small molecules can move through the gap

junctions. Of interest, other organelles were found within IP-TNTs

including endoplasmic reticulum, vesicles, and mitochondria, which

can move along the IP-TNT (Alarcon-Martinez et al., 2020), but are not

transferred to the distal pericyte because gap junctions at this interface

limit their passage.

How do IP-TNTs work? Using two-photon laser scanning microscopy,

Alarcon-Martinez et al. demonstrated that capillary pairs connected by

IP-TNTs always exhibit simultaneous, but opposite, responses triggered

by light stimulation: one capillary dilates while the other constricts

(Alarcon-Martinez et al., 2020). In other words, when the proximal

capillary dilates, the distal capillary constricts, and vice versa. Consis-

tent with caliber changes, IP-TNT-linked capillaries display opposite

blood ow responses: the dilating capillary increases blood ow, while

the constricting capillary reduces blood ow (Alarcon-Martinez et al.,

2020). Critically, targeted laser-induced ablation of individual IP-TNTs

completely impaired coordinated light-evoked changes in capillary

diameter and blood ow. Interestingly, matched responses between

IP-TNT-linked pericytes and capillaries was shown to be achieved

through intercellular Ca

waves (ICWs). Live imaging of Ca

tran-

sients in mice expressing the Ca

sensor GCaMP6 selectively in peri-

cytes, demonstrated spontaneous synchronous Ca

increases in

IP-TNT-coupled pericytes that propagated bidirectionally along IP-TNTs

(Alarcon-Martinez et al., 2020), similar to ICWs described in other TNTs

(Leybaert and Sanderson, 2012; Osswald et al., 2015). In addition,

light-evoked capillary dilation and constriction were coupled to oppo-

site and synchronized Ca

transients in pericytes connected by

IP-TNTs. ICW frequency was substantially reduced after administration

of gap junction blockers (Alarcon-Martinez et al., 2020), which is

consistent with the observation that IP-TNTs connect with distal peri-

cytes via gap junctions, known to be required for ICW propagation

(Wang et al., 2010; Sherer, 2013). Furthermore, laser-mediated ablation

of IP-TNTs disrupted ICWs and abolished capillary responses as well as

blood ow regulation after light stimulation (Alarcon-Martinez et al.,

2020). Collectively, these ndings identify IP-TNTs as novel

nanotube-like processes that are essential for pericyte-to-pericyte

communication and NVC regulation in the living retina.

The discovery of IP-TNTs challenges previous simplistic models in

which light triggers only dilation to increase blood ow. Instead, the

current data support that IP-TNTs can actively participate in the com-

plex spatial and temporal heterogeneity of blood redistribution within

the retinal capillary network. It is plausible that IP-TNTs also regulate

blood ow and NVC within the ONH and RGC targets in the brain, given

their presence in higher visual centers (Alarcon-Martinez et al., 2020),

but this remains to be demonstrated. IP-TNTs are in close contact with

neurons and glia suggesting that IP-TNTs can be directly modulated by

vasoactive factors derived from these cells. Multiple lines of evidence

argue against IP-TNTs being merely empty sleeves from regressing

vessels or bridging pericytes (Franco et al., 2015; Korn and Augustin,

2015; Watson et al., 2017; Mendes-Jorge et al., 2012), and rather sup-

port their similarity with close-ended TNTs including: i) lack of endo-

thelial cell markers, ii) expression of

-SMA, iii) presence of organelles

including motile mitochondria, iv) process stability, and v) communi-

cation through bidirectional ICWs (Alarcon-Martinez et al., 2020). Many

questions remain regarding the functional role of IP-TNTs during

development, health, and disease. The next section discusses current

data on the role of IP-TNTs during ischemic and glaucomatous damage.

Fig. 3. Structure of inter-pericyte tunneling nanotubes (IP-TNTs). Schematic

representation of an IP-TNT, a highly specialized nanotube-like process that

connects two pericytes located on distant capillary systems. An individual IP-

TNT emerges from the soma of a parent pericyte, named proximal pericyte,

and extends to a neighboring capillary where it establishes a connection with

the process of a distal pericyte. IP-TNTs are tubular with an average diameter of

~500 nm, which can extend between 4

m and 90

m in length, and contain

organelles including abundant mitochondria. Created with BioRender.com.

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

4.3. Pericytes and IP-TNTs during glaucomatous stress

In physiological conditions, pericytes and IP-TNTs play a critical in

vivo role in the regulation of local microvessel dynamics and NVC in the

adult retina (Alarcon-Martinez et al., 2020). However, what happens

when the system is under glaucomatous-related stress? Previous studies

demonstrated that pericytes can constrict capillaries in conditions of

stroke and ischemia, thus impairing capillary dynamics and preventing

blood ow during reperfusion (Yemisci et al., 2009; O’Farrell et al.,

2017; Alarcon-Martinez et al., 2019). More recently, live imaging

studies showed that pericytes constrict capillaries during ocular hyper-

tension, decreasing blood supply and compromising RGC function

(Alarcon-Martinez et al., 2022). Indeed, a substantial reduction in

capillary diameter and blood ow at pericyte locations was detected in a

mouse model of microbead-induced IOP elevation in mice, prior to overt

RGC death (Alarcon-Martinez et al., 2022). Furthermore, two-photon

microscope live imaging of capillary dynamics and blood ow, recor-

ded before and after light stimulation, showed that the ability of retinal

capillaries to dilate in response to light was severely compromised

during glaucomatous damage (Alarcon-Martinez et al., 2022). Of in-

terest, the probability of capillary blood ow blockage at pericyte lo-

cations also increased several-fold in eyes subjected to ocular

hypertension relative to sham-operated controls (Alarcon-Martinez

et al., 2022).

Consistent with their role in NVC, pathological stress has been shown

to provoke structural and functional alterations in IP-TNTs. Recent work

reported that a substantial number of IP-TNTs, both in the retina and

optic nerve, were ruptured in conditions of elevated IOP (Alarcon--

Martinez et al., 2022). A similar loss of IP-TNT integrity was observed

during ischemia or glucose deprivation (Alarcon-Martinez et al., 2020).

In these studies, IP-TNT breakage was accompanied by a substantial loss

of light-evoked capillary responses, notably impaired dilation, the

inability to regulate blood ow, and a substantial decrease in the fre-

quency of ICWs. There is now increasing evidence that Ca

homeostasis

in pericytes plays a critical role in the maintenance of IP-TNT structure

and function. In addition to playing a key role in pericyte-to-pericyte

communication through ICWs, cytosolic Ca

regulates the contractile

activity of pericytes (Burdyga and Borysova, 2018). Using transgenic

mice that express the Ca

sensor GCaMP6f selectively in pericytes, a

recent study demonstrated that ocular hypertension induced a robust

and sustained increase of Ca

within pericytes and their IP-TNTs

(Alarcon-Martinez et al., 2022). Ca

increase in pericytes was also

observed after transient ischemia and correlated with reduced frequency

of ICWs in IP-TNT-coupled pericytes (Alarcon-Martinez et al., 2020). A

major pathway of extracellular Ca

entry into pericytes is through

L-type VDCCs (Borysova et al., 2013), with the alpha 1C subunit

Fig. 4. IP-TNTs form a network of connected capillaries in all vascular plexuses of the retina and in the optic nerve. (A) Visualization of IP-TNTs in retinas from mice

expressing red uorescent protein under the control of the NG2 (Cspg4) promoter (NG2-DsRed) for selective imaging of retinal pericytes. Laminin (green) is used to

label capillaries. In healthy retinas, IP-TNTs (white arrowheads) form networks linking pericytes on separate capillary systems. Endogenous DsRed within the IP-TNT,

conrming its pericyte origin, can be seen at higher magnication (inset A

′

). (B) Analysis of the optic nerve head (ONH) in an NG2-DsRed mouse shows a network of

IP-TNTs connecting pericytes between capillaries (white arrowheads). Endogenous DsRed uorescence in the IP-TNT is shown in the higher magnication inset (B

′

(C) IP-TNTs are detected in the visual cortex and can be visualized in xed tissue or by live imaging using two-photon laser scanning microscopy (TPLSM). Modied

from (Alarcon-Martinez et al., 2020, 2021).

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Progress in Retinal and Eye Research 97 (2023) 101217

(Cav1.2) of these Ca

channels particularly enriched in pericytes

(Vanlandewijck et al., 2018). Importantly, pericyte-specic conditional

deletion of the gene encoding Cav1.2 (Cacna1c) had several benecial

effects during ocular hypertension including: i) protection of IP-TNTs, ii)

restoration of capillary dynamics, blood blow regulation, and NVC, iii)

recovery of RGC function, and iv) enhancement of RGC survival (Alar-

con-Martinez et al., 2022). Collectively, these ndings indicate that

maintaining Ca

homeostasis in pericytes is critical to restore vascular

and neuronal function in glaucoma and prevent RGC death. In addition,

these studies identify pericytes and IP-TNTs as central components of the

NVU with therapeutic potential to prevent or reverse vascular dysre-

gulation in glaucoma.

4.4. RGC-pericyte crosstalk: the chicken or the egg?

The traditional view of neurovascular interactions in glaucoma is

that, as RGCs die, there is less demand for oxygen and energy leading to

reduced local blood ow in areas of neurodegeneration. This model is

supported by studies in retina and brain showing that neurons initiate

local neurovascular response by signaling to blood vessels, directly or

indirectly, as discussed in previous sections. In this scenario, unbalanced

delivery of neuronal-derived products upon pericyte receptors may lead

to abnormal vascular function and blood delivery that will, in turn, seal

the fate of an injured neuron. Recent evidence, however, suggests that

reverse signaling from pericytes/capillaries to RGCs may also contribute

to NVC pathology in glaucoma. In this regard, a recent study used

multiphoton microscopy to longitudinally capture single-RGC Ca

re-

sponses along with blood ow in the capillary serving the same neuron

before and after pericyte-induced vessel constriction (Alarcon-Martinez

et al., 2022). This work demonstrated that when retinal capillary blood

ow was within the normal range, light-evoked RGC Ca

responses

were robust and decayed rapidly; but when blood ow was compro-

mised, Ca

signals were reduced and recovery was signicantly

delayed (Alarcon-Martinez et al., 2022). These ndings suggest that

pericyte/capillary dysfunction directly impairs neuronal activity and

may compromise RGC viability in glaucoma. In line with this reversal of

roles, the hemodynamic response has been shown to inuence neuronal

activity through mechanical, thermal, or chemical effects on astrocytes,

also known as the hemo-neural hypothesis or vasculo-neuronal coupling

(Kim et al., 2016; Moore and Cao, 2008). For example, when the

transmural pressure/ow in penetrating arterioles was increased in

brain slices, a concomitant suppression of pyramidal cell activity was

observed through activation of mechanosensitive TRPV4 channels in

brain astrocytes (Kim et al., 2016). Collectively, these data suggest an

autoregulatory mechanism in which intravascular pressure, and poten-

tially eye pressure through vascular responses, could directly modulate

neuronal activity. The implications and mechanisms underlying these

observations require further investigation.

5. Alterations of blood-retinal/brain barriers in glaucoma

In addition to being essential for the regulation of NVC in the retina

and optic nerve, the cells of the NVU play critical roles in maintaining

the integrity of the blood-retina- and blood-brain-barriers (BRB and

BBB, respectively) (O’Leary and Campbell, 2023; Moyaert et al., 2023).

The microenvironment in the retina and optic nerve is tightly regulated

and kept separate from the systemic circulation by the BRB and the BBB,

which prevent the entry of cytotoxic plasma components, blood cells,

and pathogens (Montagne et al., 2017). At the same time, these barriers

nely regulate the transport of molecules to maintain a tightly

Fig. 5. IP-TNTs connect two bona de pericytes via CX43 gap junctions. (A) Electroporation of a single pericyte with the low molecular weight marker uorescein

shows rapid diffusion of the dye from the soma and IP-TNT into the distal pericyte demonstrating a direct connection between IP-TNT-linked pericytes (pipette shown

in green). (B) When uorescein is electroporated in the presence of the gap junction blocker carbenoxolone (CBX), the dye accumulates in the proximal pericyte and

IP-TNT delineating the contact between the IP-TNT end-foot (ef) shown in green, and the distal pericyte process (dpp) shown in red (see insets). Fluorescein

accumulation in the proximal pericyte allows unmistakable identication of the connection between the IP-TNT terminal and the distal pericyte. (C) Correlative serial

block-face focused ion beam scanning electron microscopy (FIB-SEM) revealed that IP-TNT end-feet (ef) connect with the distal pericyte process (dpp) through direct

membrane-to-membrane contacts, evocative of gap junctions (white arrowheads). (D) Consistent with this, connexin 43 (Cx43) is detected in IP-TNT terminals (end-

foot: ef) in Cx43-ECFP reporter mice (white arrowheads). Lectin is used to label pericytes, IP-TNTs, and endothelial cells. Modied from (Alarcon-Martinez

et al., 2020).

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

controlled milieu that is required for optimal tissue homeostasis and

neuronal activity (Zhao et al., 2015b). The outer BRB consists of the

choroid, Bruch’s membrane, and retinal pigment epithelium, while the

inner BRB is demarcated by the endothelial cells that line the retinal

vasculature originating from the retinal artery. The properties of the

inner BRB are dened by the cells of the NVU, of which pericytes,

endothelial, and glial cells play key roles (Sweeney et al., 2019), and will

be discussed in subsequent sections. For the purpose of this review and

in the context of glaucoma, the term BRB will be used solely for the inner

BRB. The vascular barrier at the ONH and optic nerve is an intrinsic part

of the central nervous system and thus considered very similar in

structure and function to the BBB (Nian et al., 2021). In disease states,

including glaucoma, BRB/BBB breakdown can result in the leakage of

harmful blood components into the retina and optic nerve leading to

inammation, cellular inltration, aberrant transport and clearance of

molecules, reduced blood ow, and neuronal decits (Zhao et al.,

2015b; O’Leary and Campbell, 2023). In the following sections, we will

cover the clinical evidence supporting alterations of the BRB/BBB

function in glaucoma patients as well as cellular and molecular mech-

anisms involved in the regulation of vascular permeability.

5.1. Loss of vascular integrity in glaucoma

5.1.1. Fluorescein angiography studies

Accumulating evidence from pre-clinical and clinical studies using

uorescein, a recognized physiological marker of the integrity of the

BRB/BBB function (Cunha-Vaz, 2004), suggest vascular barrier disrup-

tion in glaucoma. Experimental IOP elevation in non-human primate

eyes demonstrated uorescein extravasation at the optic disc (Radius

and Anderson, 1980). A number of clinical studies also reported uo-

rescein lling defects and increased leakage at the optic disc in glau-

coma relative to normotensive individuals (Arend et al., 2004, 2005;

Ashworth and Rosen, 1970; Plange et al., 2006, 2010, 2012; Schwartz,

1994; Schwartz et al., 1977; Spaeth, 1975; Tsukahara, 1978). Fluores-

cein dye extravasation is a dynamic process and, as such, the relative

increase of the late phase of uorescein leakage may suggest higher

vascular permeability at the optic nerve, which could stem from damage

to the BBB (Anderson and Davis, 1996; Orgül et al., 1999). For example,

the evaluation of the time course of uorescein leakage of the ONH in

glaucoma patients revealed an increase from 7–8 min to 9–10 min,

which was signicantly higher relative to healthy controls (Plange et al.,

2010). In addition, Arend et al. identied greater leakage in glaucom-

atous eyes using a semiquantitative digital image analysis in late-phase

angiogram (Arend et al., 2005). Leakage worsened in areas of peri-

papillary choroidal atrophy, a well-known feature of glaucomatous optic

neuropathy (Schwartz, 1994; Schwartz et al., 1977; Spaeth, 1975; Tsu-

kahara, 1978). Using uorescein angiography with a scanning laser

ophthalmoscope and semi-quantied digital image analysis, Plange

et al. identied increased uorescein leakage at the optic disc in patients

with high IOP (Plange et al., 2010). However, no association was found

with the extent of glaucomatous damage, either in visual eld damage or

cup-disc ratios (Plange et al., 2010), suggesting that glaucomatous

uorescein leakage may occur spatially and temporally regardless of

disease severity.

Fluorescein angiography has also revealed prolonged arteriovenous

transit times, with frequent vessel lling defects or delayed lling in

glaucomatous eyes with disc hemorrhages, suggesting vascular stasis

and thrombus formation at disc hemorrhage sites as a consequence of

vascular leakage (Park et al., 2015). Absolute and relative uorescein

lling defects of the ONH have been reported in glaucomatous eyes

(Schwartz et al., 1977; Spaeth, 1975; Talusan and Schwartz, 1977).

Absolute lling defects are larger and more frequent in patients with

glaucoma compared with healthy controls and correlate with capillary

dropout in the surface nerve ber layer of the optic disc (Loebl and

Schwartz, 1977; Nanba and Schwartz, 1988; Schwartz et al., 1977).

Fluorescein lling defects are preferentially located at the margin of the

optic disc excavation, predominantly at inferotemporal and super-

otemporal locations, and are often found at the wall rather than at the

oor of the cup (Adam and Schwartz, 1980; Fishbein and Schwartz,

1977; Schwartz, 1994; Schwartz et al., 1977; Arnold, 1995). The relative

defects are characterized by delayed uorescence, and they are inter-

preted as areas of hypovascularity. A correlation between the number,

extent, and topography of uorescein lling defects with visual eld

loss, retinal nerve ber layer defects, and excavation of the disc has been

reported in glaucoma patients (Nanba and Schwartz, 1988; Talusan and

Schwartz, 1977; Tsukahara, 1978).

5.1.2. Disc hemorrhages

An association between glaucoma and optic disc hemorrhages was

rst described in 1876 by Albert Emmerich, who reported bleeding

within the ONH and retina in some glaucoma patients (Emmerich,

1876). It was not until 1970 that Stephen Drance and Ian Begg found an

association between disc hemorrhages and visual eld defects, sug-

gesting that they are important pathological features in individuals

affected by glaucoma (Drance and Begg, 1970; Begg et al., 1970). Disc

hemorrhages are small splinter-like or ame-shaped areas of bleeding

located in the neuroretinal rim of the optic disc and the adjacent su-

percial nerve ber layer (Drance, 1989). Accumulating clinical evi-

dence conrms that optic disc hemorrhages are a signicant risk factor

for the development and progression of glaucoma and can be predictive

of future damage in patients with both primary open-angle and

low-tension glaucoma (Budenz et al., 2017; Jasty et al., 2020).

The disruption of the integrity of the capillary wall at the ONH is

considered to be at the origin of disc hemorrhages. Although the

mechanisms underlying the formation of disc hemorrhages are still

unknown, three main hypotheses have been proposed including

vascular defects, biomechanical stress, and reactive gliosis (Jasty et al.,

2020). Vascular dysregulation and alterations in NVU components can

lead to abnormal vessel constriction, microinfarction in the optic disc,

and decreased capillary perfusion (Drance and Begg, 1970; Grieshaber

et al., 2007; Lichter and Henderson, 1978), which increase the vulner-

ability of capillaries to rupture. In addition, the release of vasoactive

agents, cytokines, and mediators that increase BRB/BBB permeability

such as ET-1 and metalloproteinase-9 (MMP-9) may weaken the base-

ment membrane surrounding the capillary walls resulting in leaky ves-

sels (Mi et al., 2012; Lee et al., 2014). Microvascular ischemia and

autoregulatory dysfunction have also been suggested to play a role in the

development of disc hemorrhages (Grieshaber et al., 2006). Biome-

chanical strain, including sheer and compression forces, are known to

cause deformation of the lamina cribrosa as well as the prelaminar rim

tissue and can lead to microvascular disruption, leaky capillaries,

disturbance of the BRB/BBB, and hemodynamic defects (Quigley et al.,

1981, Burgoyne et al., 2005; Kim et al., 2015; Lee et al., 2022). Posterior

stress/strain forces at the lamina cribrosa may also affect vascular

endothelial cell differentiation, maturation, and molecular signaling

which may alter endothelial cell morphology, gene expression, and

function (Akimoto et al., 2000; Ott et al., 1995). Lastly, proliferative

reactive gliosis has been proposed to promote glial scar formation, hence

remodeling the ONH environment, causing breakdown of capillary walls

that result in optic disc hemorrhages (Lee et al., 2017a). Consistent with

this, reactive astrocytes produce several mediators known to open the

BRB/BBB including ATP, glutamate, aspartate, taurine, and

macrophage-inammatory protein 2 (Abbott, 2002; Chen et al., 2000;

Kustova et al., 1999). A better understanding of the origin of disc

hemorrhages may provide insights into the role of vascular leakage in

glaucoma.

5.2. Pericytes and the regulation of vascular permeability

In addition to the critical role of pericytes in the control of capillary

blood ow and NVC discussed in previous sections, these cells are

crucial for the maintenance of vascular barriers and their integrity

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

(Armulik et al., 2010; Daneman et al., 2010; Bell et al., 2010). Pericytes

are required to develop and maintain the homeostasis of the BRB/BBB

(Trost et al., 2016; O’Leary and Campbell, 2023). For example, pericytes

are a key source of basement membrane proteins at the BRB/BBB such as

laminins and vitronectin (Yao et al., 2014; He et al., 2016) and they are

important players in endothelial tube formation and stabilization

(Armulik et al., 2011). During vascular development, pericytes are

recruited by platelet-derived growth factor B (PDGF-B), produced by

endothelial cells, which activates the PDGF receptor-β (PDGFR-β) on

pericytes (Park et al., 2017). Blockade of PDGF-B/PDGFRβ signaling in

mice using multiple approaches including conditional deletion of the

PDGFB gene (Enge, 2002; Park et al., 2017) or its retention motif

(Lindblom et al., 2003) and administration of PDGFRβ blocking anti-

bodies (Uemura et al., 2002; Ogura et al., 2017) resulted in vascular

barrier disruption characterized by reduced pericyte coverage,

increased BRB/BBB permeability, and microaneurysms (Armulik et al.,

2010; Daneman et al., 2010; Bell et al., 2010; Lindahl et al., 1997).

Analysis of mice with different combinations of null, hypomorphic, and

wild-type PDGFRβ alleles showed that the permeability of the BBB

correlated inversely with the amount of pericyte vessel coverage,

demonstrating that the number of pericytes directly determines vessel

permeability during development (Daneman et al., 2010).

In the retina, inhibition of pericyte recruitment using a PDGFRβ

blocking antibody promoted the dissociation of pericytes from endo-

thelial cells and BRB breakdown in adult mice reproducing classical

features of diabetic retinopathy such as increased vessel leakage,

hypoperfusion, and angiogenesis (Ogura et al., 2017). In this study,

transcriptomic analysis of pericyte-depleted endothelial cells showed

upregulation of inammatory genes, which correlated with an inux of

inltrating leukocytes intimately associated with the denuded vessels

(Ogura et al., 2017). Tamoxifen-induced conditional deletion of PDGFB

from developing endothelial cells conrmed that impaired pericyte

recruitment to retinal vessels leads to BRB disruption and diabetic

retinopathy-like features (Park et al., 2017). Despite retinal pericyte

loss, however, tamoxifen treatment in adult mice did not cause BRB

disintegration unless endothelial cells were challenged with exogenous

vascular endothelial growth factor-A (VEGF-A), which led to upregula-

tion of angiopoietin-2 (Ang2) and BRB breakdown (Park et al., 2017).

Thus, in addition to PDGF-B/PDGFRβ signaling, the interaction between

pericytes and endothelial cells involves other pathways including

Ang2/Tie-2 receptor signaling, transforming growth factor-β (TGF-β),

and Notch receptors and their ligands (Sweeney and Foldes, 2018).

These ndings support a tight pericyte-endothelial cell crosstalk in the

regulation of BRB/BBB integrity that involves paracrine mediators as

well as changes in gene expression (Geevarghese and Herman, 2014;

M¨

ae et al., 2021).

BRB/BBB breakdown secondary to pericyte depletion eventually

leads to neuronal dysfunction and functional cognitive impairment in

prevalent pathologies such as diabetic retinopathy and Alzheimer’s

disease (Yamazaki and Kanekiyo, 2017; Uemura et al., 2020; O’Leary

and Campbell, 2023; Nikolakopoulou et al., 2019). However, there is no

evidence of pericyte death in glaucoma suggesting that other mecha-

nisms of pericyte dysfunction can potentially contribute to BRB/BBB loss

of integrity in this disease. A number of mediators could participate in

pericyte-induced BRB/BBB breakdown. For example, using a model of

ischemic damage induced by photothrombotic occlusion of cortical

capillaries, Underly and colleagues showed that capillary leakage ap-

pears at sites where pericyte soma are in contact with endothelial cells

(Underly et al., 2017). Plasma leakage during cerebral ischemia corre-

lated with MMP activation in pericytes, detected using a FITC-gelatin

probe MMP-2/9 activity (Underly et al., 2017). Injection of a MMP-9

inhibitor reduced pericyte-associated FITC-gelatin uorescence and

plasma leakage in the BBB (Underly et al., 2017). In the retina, MMP-9

activity has long been linked to RGC death in several models of retinal

damage, including ocular hypertension (Guo et al., 2005), optic nerve

transection (Sun et al., 2011), optic nerve ligation (Zhang et al., 2004b;

Zhang and Chintala, 2004), and kainic acid excitotoxicity (Zhang et al.,

2004a). Whether pericytes contribute to glaucoma pathophysiology by

BRB/BBB dysfunction caused by MMP proteolytic degradation or by

other mechanisms remains unknown.

5.3. Tight and adherens junctions in endothelial cells

Unlike the leaky capillary endothelium in peripheral organs, endo-

thelial cells that line the BRB/BBB are sealed by tight junctions and have

low rates of transcytosis (Reese and Karnovsky, 1967; Tietz and Engel-

hardt, 2015; Siegenthaler et al., 2013; Daneman and Prat, 2015).

Indeed, endothelial cells with tight and adherens junctions are a major

barrier keeping the retinal microenvironment closed off from the gen-

eral circulation (Nitta et al., 2003; Fresta et al., 2020). Tight junctions,

located near the endothelial cell apical membrane, include claudins (1,

3, 5, and 12), occludin, and junctional adhesion molecules, which limit

the paracellular diffusion of ions and solutes (Tietz and Engelhardt,

2015). Claudin-5 is a critical determinant of BBB/BRB permeability, and

loss of claudin-5 function has been shown to increase vascular leakage in

both retina and brain (Nitta et al., 2003; Argaw et al., 2009; Tian et al.,

2014; Muthusamy et al., 2014; Greene et al., 2019). Downregulation of

claudin-5 was reported in retinas from mice subjected to hypoxia, which

correlated with increased BRB permeability, similar to the phenotype

seen in claudin-5 decient mice (Koto et al., 2007). Claudin-5 down-

regulation was reported in a mouse model of acute ocular hypertension

(Zhao et al., 2020). Claudins can also be used therapeutically to seal the

BBB as shown in a model of experimental autoimmune encephalomy-

elitis by increasing the expression of claudin-1 (Pfeiffer et al., 2011).

Tight junction proteins are linked to the actin and vinculin-based cyto-

skeleton through scaffolding zona occludens proteins of the

membrane-associated guanylate kinase family (ZO-1, -2, and -3) (Tor-

navaca et al., 2015). ZO-1 deciency can contribute to BBB breakdown

in chronic and acute neurodegenerative disorders (Zlokovic, 2011). Of

interest, pericyte deciency or dysfunction can cause a reduction in the

expression of tight junction proteins by endothelial cells including

claudin-5, occludin, and ZO-1 (Bell et al., 2010), which increase ow

transcytosis across vascular barriers (Armulik et al., 2010).

The adherens junction proteins, vascular endothelial-cadherin (VE-

cadherin) and platelet endothelial cell adhesion molecule-1 (PECAM-1),

form homophilic cell-to-cell contacts near the basolateral membrane of

endothelial cells (Vorbrodt and Dobrogowska, 2003; Dejana and Vest-

weber, 2013; Tietz and Engelhardt, 2015). Adherens junctions connect

to the cytoskeleton, modulate receptor signaling, and regulate the

trans-endothelial passage of immune cells including monocytes, neu-

trophils, and lymphocytes (Giri et al., 2000; Allport et al., 2000; Tur-

owski et al., 2008; Wessel et al., 2014). In the retina, loss of VE-cadherin

was observed in mouse retinal organotypic cultures exposed to elevated

eye pressure (Brockhaus et al., 2020). However, the phosphorylation of

VE-cadherin at tyrosine residues was shown to be required for the

regulation of trans-endothelial migration of leukocytes in the brain

(Turowski et al., 2008; Wessel et al., 2014). Therefore, in addition to

changes in gene and protein expression, the analysis of VE-cadherin

phosphorylation would provide useful information about its contribu-

tion to vascular leakage in glaucoma. Overall, the functional role of tight

and adherens junctions in the onset and progression of BRB/BBB

breakdown in glaucoma remains to be elucidated.

5.4. Connexins and pannexins

Connexins and pannexins are transmembrane proteins that allow the

exchange of ions and molecules allowing autocrine and paracrine

signaling between cells. Connexin proteins form both connexin gap

junction channels and hemichannels. While gap junctions are required

for a number of physiological and neuroprotective functions, increasing

evidence suggests that hemichannels exert a pathological and pro-

degenerative role during injury or disease (Danesh-Meyer et al., 2016;

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

Boal et al., 2021). Connexin gap junction channels mediate cell-to-cell

intercellular communication and signaling (Willebrords et al., 2016)

by facilitating the passive diffusion of ions and metabolites between

adjacent cells to regulate electrical and chemical transmission (Weid-

mann, 1969). Connexin hemichannels, on the other hand, are cyto-

plasmic non-selective membrane pores that allow the passage of

molecules up to ~1 kDa in size (Yang et al., 2023). Due to their poor

selectivity and massive opening, which allows the passage of toxic me-

tabolites, the function of connexin hemichannels has been associated

with pathological conditions (Guo et al., 2016). Connexin hemichannels

are considered pathological pores primarily due to their low opening

probability in physiological conditions and large opening probability in

response to cellular stress (Contreras et al., 2003; Lander et al., 1996;

Giaume et al., 2013; Acosta et al., 2021). Consequently, dysregulated

connexin hemichannel opening has been shown to promote cell death

because of loss of osmoregulation, excitotoxicity, and inammation

(Yang et al., 2023).

Pannexins are also cytoplasmic channels permeable to small mole-

cules including Ca

, glutamate, and ATP, but they differ from con-

nexins in that they rarely form intercellular channels (Cotrina et al.,

1998; Wei et al., 2016b). Although pannexins and connexins are struc-

turally similar, they do not share enough sequence similarity to have a

common ancestry (Beyer and Berthoud, 2018). The mechanisms that

trigger connexin hemichannel opening can also activate pannexin

channels including mechanical stimulation and Ca

increase (Wei

et al., 2016b; Cotrina et al., 1998; Lohman and Isakson, 2014). Pannexin

channels are self-regulated and they close in response to extracellular

ATP, an important trigger during retinal injury (Barbe et al., 2006).

Recent evidence highlights the time-dependent roles of connexin hem-

ichannel and pannexin channels during ischemia (Mugisho et al., 2018).

It has been proposed that pannexin channels initiate the early response

to ischemic injury, and connexin hemichannels perpetuate the response

in sustained pathological conditions (Bernstein and Fishman, 2016;

Davidson et al., 2013).

Connexin 43 (CX43) is the most ubiquitous of all connexins and plays

an important role in several retinal injuries and diseases (Danesh-Meyer

et al., 2016; Boal et al., 2021). CX43 hemichannels are present in

endothelial cells, pericytes, Müller cells/astrocytes in both retina and

optic nerve of mice and humans (Janssen-Bienhold et al., 1998; Kerr

et al., 2010), thus they are key elements for crosstalk within the NVU.

IP-TNTs in the retina connect distal pericytes through their specialized

endfeet processes rich in CX43, which is essential for Ca

-based

communication (Alarcon-Martinez et al., 2020). CX43 expression has

been shown to increase in an organotypic model of optic nerve ischemia,

and antisense oligonucleotide treatment substantially reduced CX43

levels and the ensuing inammatory response (Danesh-Meyer et al.,

2008). Partial optic nerve transection evoked a biphasic CX43: rapid

upregulation in the retinal area corresponding to axonal damage and

delayed elevation in the unaffected area, which coincided with signi-

cant RGC loss (Chew et al., 2011). A study of human post-mortem eyes

demonstrated a signicant increase in CX43 labeling in the lamina cri-

brosa of glaucoma patients (Kerr et al., 2010, 2011). In response to

cellular stress, CX43 hemichannels mediate ATP release into the extra-

cellular space (Cotrina et al., 1998; Iglesias et al., 2009; Liu et al., 2008),

which activates the purinergic inammatory pathway by binding to the

purinergic receptor, P2RX7 (Calder et al., 2015; Cotrina and Neder-

gaard, 2009; Huang et al., 2012). Extracellular ATP release via CX43

hemichannels was conrmed using CX43 knockout mice in which this

response was abrogated (Huang et al., 2012).

Accumulating evidence support a role for CX43 hemichannels in

vascular leakage in the retina and optic nerve. Mechanical stress and

strain has been shown to increase CX43 hemichannel expression leading

to ATP release from ONH astrocytes (Bao et al., 2004; Beckel et al., 2014;

Dubyak, 2009). In a retinal ischemia-reperfusion injury model, treat-

ment with the non-specic gap junction channel blocker carbenoxolone,

the hemichannel blocker lanthanum chloride, or CX43 hemichannel

mimetic blocking peptides prevented endothelial cell death, restored

vascular integrity, and promoted RGC survival (Danesh-Meyer et al.,

2012). These results were conrmed ex vivo in preparations of rat and

human microvascular endothelial cells exposed to hypoxia and treated

with CX43 blocking peptides (Danesh-Meyer et al., 2012) used at a

concentration that blocks hemichannels but does not uncouple gap

junctions (O’Carroll et al., 2008). In this study, endothelial cell

dysfunction was mediated by opening of CX43 hemichannels in endo-

thelial cells in the absence of astrocytes (Danesh-Meyer et al., 2012).

This nding suggests that ischemia can cause CX43

hemichannel-mediated loss of endothelial cell integrity without the

involvement of astrocytes. High pressure-induced retinal

ischemia-reperfusion also resulted in upregulation of CX43 expression in

endothelial cells and astrocytes in the retinal nerve ber layer, which

correlated with BRB breakdown (Kerr et al., 2012). The role of CX43 in

BRB breakdown was conrmed using a CX43 hemichannel blocking

peptide, which decreased BRB disruption and neuroinammation (Chen

et al., 2015; O’Carroll et al., 2013). Collectively, these ndings suggest

that the upregulation of CX43 hemichannels induced by ischemic injury

is an initiating event responsible for vascular leakage and an inam-

matory cascade leading to RGC death. The modulation of CX43 hemi-

channel opening has also been shown to effectively restore vascular

integrity in models of perinatal ischemia, spinal cord injury, and

light-induced macular damage (O’Carroll et al., 2008; Guo et al., 2016;

Mao et al., 2017; Galinsky et al., 2018; Mat Nor et al., 2020). These

studies open up the possibility of using strategies to modulate CX43

function to prevent BRB/BBB breakdown and protect RGCs from

ensuing damage.

6. Conclusions and future directions

Accumulating evidence supports a connection between neuro-

vascular dysfunction and visual decits in glaucoma patients. The na-

ture of vascular defects is complex and includes impaired

autoregulation, NVC dysfunction, and loss of BRB/BBB integrity. An

imbalance in the regulation of microcirculatory blood ow and vascular

breakdown can increase the susceptibility of vulnerable RGCs to

stressors leading to neurodegeneration. Cell-cell interactions within

members of the NVU is critical for hemodynamic regulation and barrier

maintenance. Recent data suggest that signaling between NVU cells is

altered in glaucoma, but our current understanding of these processes

and how they relate to RGC dysfunction is still in its infancy. Due to their

strategic location, pericytes are emerging as key players in the regula-

tion of blood ow and NVC in the retina and optic nerve both in phys-

iological and glaucomatous conditions. Pericytes communicate with

each other via IP-TNTs, which provide a physical nexus for the coordi-

nation of light-evoked blood ow within a local capillary network. IP-

TNT damage triggered by ocular hypertension results in loss of NVC

and capillary responses, while strategies that protect IP-TNTs restore

blood ow promoting RGC survival and functional recovery. A better

understanding of how pericytes interact with other cells of the NVU will

provide insights into the causes of NVC dysfunction and will expand the

identication of potential therapeutic targets to restore hemodynamic

homeostasis in glaucoma. Unlike neurons, which have a limited capacity

for regeneration, vascular cells are plastic and contain self-repair

mechanisms thus presenting a unique opportunity to develop strate-

gies to support neurovascular function and maintain RGC health.

Author statement

LAM: conceptualization, data curation, formal analysis, investiga-

tion, methodology, writing - original draft, visualization; YS: data

curation, formal analysis, investigation, methodology, writing - original

draft, visualization; DVB: data curation, formal analysis, investigation,

methodology, writing - original draft, visualization; JLCV: writing -

original draft; IAVP: writing - original draft; HQ: writing - original draft,

L. Alarcon-Martinez et al.

Progress in Retinal and Eye Research 97 (2023) 101217

visualization; BF: writing - original draft, HDM: writing - original draft;

ADP: conceptualization, data curation, formal analysis, writing – orig-

inal draft, funding acquisition, supervision. All authors: reviewing and

editing of the manuscript.

Permission was obtained for the use of copyrighted gures from

other sources. The reference is included in the gure legend.

Declaration of competing interest

The authors declare that they have no competing nancial interest

regarding the content of this work.

Data availability

Data will be made available on request.

Acknowledgements

We thank Dr. Lin Wang for valuable comments on the manuscript.

Funding: Canadian Institutes of Health Research (ADP), National In-

stitutes of Health (ADP, BF), Glaucoma Foundation (ADP), BrightFocus

Foundation (ADP), Alcon Research Institute (ADP, LAM), and Australian

Vision Research (LAM), Perpetual IMPACT (LAM), and Fighting Blind-

ness Canada (LAM, ADP). ADP and LAM are Alcon Research Institute

Senior Investigators. ADP holds a Canada Research Chair (Tier 1). YS

was supported by postdoctoral fellowships from the Fonds de recherche

Sant´

e – Qu´

ebec (FRQS) and CIHR, DVB was supported by a graduate

scholarship from FRQS, HQ was supported by a postdoctoral fellowship

from the Mexican National Council of Science and Technology

(CONACYT).

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Jun 2023

Purpose: Tissue stiffening and alterations in retinal blood flow have both been suggested as causative mechanisms of glaucomatous damage. We tested the hypothesis that retinal blood vessels also stiffen, using laser speckle flowgraphy (LSFG) to characterize vascular resistance. Methods: In the longitudinal Portland Progression Project, 231 eyes of 124 subjects received LSFG scans of the optic nerve head (ONH) and automated perimetry every 6 months for six visits. Eyes were classified as either "glaucoma suspect" or "glaucoma" eyes based on the presence of functional loss on the first visit. Vascular resistance was quantified using the mean values of several instrument-defined parameterizations of the pulsatile waveform measured by LSFG, either in major vessels within the ONH (serving the retina) or in capillaries within ONH tissue, and age-adjusted using a separate group of 127 healthy eyes of 63 individuals. Parameters were compared against the severity and rate of change of functional loss using mean deviation (MD) over the six visits, within the two groups. Results: Among 118 "glaucoma suspect" eyes (average MD, -0.4 dB; rate, -0.45 dB/y), higher vascular resistance was related to faster functional loss, but not current severity of loss. Parameters measured in major vessels were stronger predictors of rate than parameters measured in tissue. Among 113 "glaucoma" eyes (average MD, -4.3 dB; rate, -0.53 dB/y), higher vascular resistance was related to more severe current loss but not rate of loss. Conclusions: Higher retinal vascular resistance and, by likely implication, stiffer retinal vessels were associated with more rapid functional loss in eyes without significant existing loss at baseline.

Neuroprotection in glaucoma: Mechanisms beyond intraocular pressure lowering

Article

Full-text available

Jun 2023

Glaucoma is a common, complex, multifactorial neurodegenerative disease characterized by progressive dysfunction and then loss of retinal ganglion cells, the output neurons of the retina. Glaucoma is the most common cause of irreversible blindness and affects ∼80 million people worldwide with many more undiagnosed. The major risk factors for glaucoma are genetics, age, and elevated intraocular pressure. Current strategies only target intraocular pressure management and do not directly target the neurodegenerative processes occurring at the level of the retinal ganglion cell. Despite strategies to manage intraocular pressure, as many as 40% of glaucoma patients progress to blindness in at least one eye during their lifetime. As such, neuroprotective strategies that target the retinal ganglion cell and these neurodegenerative processes directly are of great therapeutic need. This review will cover the recent advances from basic biology to on-going clinical trials for neuroprotection in glaucoma covering degenerative mechanisms, metabolism, insulin signaling, mTOR, axon transport, apoptosis, autophagy, and neuroinflammation. With an increased understanding of both the basic and clinical mechanisms of the disease, we are closer than ever to a neuroprotective strategy for glaucoma.

Imaging blood-brain barrier dysfunction: A state-of-the-art review from a clinical perspective

Article

Full-text available

Apr 2023

The blood-brain barrier (BBB) consists of specialized cells that tightly regulate the in- and outflow of molecules from the blood to brain parenchyma, protecting the brain’s microenvironment. If one of the BBB components starts to fail, its dysfunction can lead to a cascade of neuroinflammatory events leading to neuronal dysfunction and degeneration. Preliminary imaging findings suggest that BBB dysfunction could serve as an early diagnostic and prognostic biomarker for a number of neurological diseases. This review aims to provide clinicians with an overview of the emerging field of BBB imaging in humans by answering three key questions: (1. Disease) In which diseases could BBB imaging be useful? (2. Device) What are currently available imaging methods for evaluating BBB integrity? And (3. Distribution) what is the potential of BBB imaging in different environments, particularly in resource limited settings? We conclude that further advances are needed, such as the validation, standardization and implementation of readily available, low-cost and non-contrast BBB imaging techniques, for BBB imaging to be a useful clinical biomarker in both resource-limited and well-resourced settings.

Pericytes: The lung-forgotten cell type

Article

Full-text available

Mar 2023

Pericytes are a heterogeneous population of mesenchymal cells located on the abluminal surface of microvessels, where they provide structural and biochemical support. Pericytes have been implicated in numerous lung diseases including pulmonary arterial hypertension (PAH) and allergic asthma due to their ability to differentiate into scar-forming myofibroblasts, leading to collagen deposition and matrix remodelling and thus driving tissue fibrosis. Pericyte-extracellular matrix interactions as well as other biochemical cues play crucial roles in these processes. In this review, we give an overview of lung pericytes, the key pro-fibrotic mediators they interact with, and detail recent advances in preclinical studies on how pericytes are disrupted and contribute to lung diseases including PAH, allergic asthma, and chronic obstructive pulmonary disease (COPD). Several recent studies using mouse models of PAH have demonstrated that pericytes contribute to these pathological events; efforts are currently underway to mitigate pericyte dysfunction in PAH by targeting the TGF-β, CXCR7, and CXCR4 signalling pathways. In allergic asthma, the dissociation of pericytes from the endothelium of blood vessels and their migration towards inflamed areas of the airway contribute to the characteristic airway remodelling observed in allergic asthma. Although several factors have been suggested to influence this migration such as TGF-β, IL-4, IL-13, and periostin, recent evidence points to the CXCL12/CXCR4 pathway as a potential therapeutic target. Pericytes might also play an essential role in lung dysfunction in response to ageing, as they are responsive to environmental risk factors such as cigarette smoke and air pollutants, which are the main drivers of COPD. However, there is currently no direct evidence delineating the contribution of pericytes to COPD pathology. Although there is a lack of human clinical data, the recent available evidence derived from in vitro and animal-based models shows that pericytes play important roles in the initiation and maintenance of chronic lung diseases and are amenable to pharmacological interventions. Therefore, further studies in this field are required to elucidate if targeting pericytes can treat lung diseases.

Pericytes and the Control of Blood Flow in Brain and Heart

Article

Full-text available

Feb 2023
ANNU REV PHYSIOL

Pericytes, attached to the surface of capillaries, play an important role in regulating local blood flow. Using optogenetic tools and genetically encoded reporters in conjunction with confocal and multiphoton imaging techniques, the 3D structure, anatomical organization, and physiology of pericytes have recently been the subject of detailed examination. This work has revealed novel functions of pericytes and morphological features such as tunneling nanotubes in brain and tunneling microtubes in heart. Here, we discuss the state of our current understanding of the roles of pericytes in blood flow control in brain and heart, where functions may differ due to the distinct spatiotemporal metabolic requirements of these tissues. We also outline the novel concept of electro-metabolic signaling, a universal mechanistic framework that links tissue metabolic state with blood flow regulation by pericytes and vascular smooth muscle cells, with capillary KATP and Kir2.1 channels as primary sensors. Finally, we present major unresolved questions and outline how they can be addressed.

Myeloid masquerade: Microglial transcriptional signatures in retinal development and disease

Article

Full-text available

Jan 2023

Microglia are dynamic guardians of neural tissue and the resident immune cells of the central nervous system (CNS). The disease-associated microglial signature (DAM), also known as the microglial neurodegenerative phenotype (MGnD), has gained significant attention in recent years as a fundamental microglial response common to various neurodegenerative disease pathologies. Interestingly, this signature shares many features in common with developmental microglia, suggesting the existence of recycled gene programs which play a role both in early neural circuit formation as well as in response to aging and disease. In addition, recent advances in single cell RNA sequencing have revealed significant heterogeneity within the original DAM signature, with contributions from both yolk sac-derived microglia as well as bone marrow-derived macrophages. In this review, we examine the role of the DAM signature in retinal development and disease, highlighting crosstalk between resident microglia and infiltrating monocytes which may critically contribute to the underlying mechanisms of age-related neurodegeneration.

Brain capillary pericytes are metabolic sentinels that control blood flow through a KATP channel-dependent energy switch

Article

Full-text available

Dec 2022

Despite the abundance of capillary thin-strand pericytes and their proximity to neurons and glia, little is known of the contributions of these cells to the control of brain hemodynamics. We demonstrate that the pharmacological activation of thin-strand pericyte KATP channels profoundly hyperpolarizes these cells, dilates upstream penetrating arterioles and arteriole-proximate capillaries, and increases capillary blood flow. Focal stimulation of pericytes with a KATP channel agonist is sufficient to evoke this response, mediated via KIR2.1 channel-dependent retrograde propagation of hyperpolarizing signals, whereas genetic inactivation of pericyte KATP channels eliminates these effects. Critically, we show that decreasing extracellular glucose to less than 1 mM or inhibiting glucose uptake by blocking GLUT1 transporters in vivo flips a mechanistic energy switch driving rapid KATP-mediated pericyte hyperpolarization to increase local blood flow. Together, our findings recast capillary pericytes as metabolic sentinels that respond to local energy deficits by increasing blood flow to neurons to prevent energetic shortfalls.

Dissecting the neurovascular unit in physiology and Alzheimer's disease: Functions, imaging tools and genetic mouse models

Article

Apr 2023
NEUROBIOL DIS

The neurovascular unit (NVU) plays an essential role in regulating neurovascular coupling, which refers to the communication between neurons, glia, and vascular cells to control the supply of oxygen and nutrients in response to neural activity. Cellular elements of the NVU coordinate to establish an anatomical barrier to separate the central nervous system from the milieu of the periphery system, restricting the free movement of substances from the blood to the brain parenchyma and maintaining central nervous system homeostasis. In Alzheimer's disease, amyloid-β deposition impairs the normal functions of NVU cellular elements, thus accelerating the disease progression. Here, we aim to describe the current knowledge of the NVU cellular elements, including endothelial cells, pericytes, astrocytes, and microglia, in regulating the blood-brain barrier integrity and functions in physiology as well as alterations encountered in Alzheimer's disease. Furthermore, the NVU functions as a whole, therefore specific labeling and targeting NVU components in vivo enable us to understand the mechanism mediating cellular communication. We review approaches including commonly used fluorescent dyes, genetic mouse models, and adeno-associated virus vectors for imaging and targeting NVU cellular elements in vivo.

Connexin Hemichannels and Pannexin Channels in Toxicity: Recent Advances and Mechanistic Insights

Article

Mar 2023
TOXICOLOGY

Connexin hemichannels and pannexin channels are two types of transmembrane channels that allow autocrine/paracrine signalling through the exchange of ions and molecules between the intra- and extracellular compartments. However, owing to the poor selectivity of permeable ions and metabolites, the massive opening of these plasma membrane channels can lead to an excessive influx of toxic substances and an outflux of essential metabolites, such as adenosine triphosphate, glutathione, glutamate and ions, resulting in unbalanced cell homeostasis and impaired cell function. It is becoming increasingly clear that these channels can be activated in response to external stimuli and are involved in toxicity, yet their concrete mechanistic roles in the toxic effects induced by stress and various environmental changes remain poorly defined. This review provides an updated understanding of connexin hemichannels and pannexin channels in response to multiple extrinsic stressors and how these activated channels and their permeable messengers participate in toxicological pathways and processes, including inflammation, oxidative damage, intracellular calcium imbalance, bystander DNA damage and excitotoxicity.

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