Mishra Lab

This image shows glial cells and blood vessels of the retina, which is the nervous tissue in the back of the eye. Tiny blood vessels called capillaries are shown in blue, vascular cells called pericytes are in red, and glial cells called astrocytes are in green. Astrocytes relay messages from active neurons to pericytes on capillaries to co-ordinate blood flow.

Astrocytes in neurovascular coupling

Side-by-side images contrasting  brain sections of the healthy rat and the post-stroke rat.
GFAP immunolabeled astrocyte endfeet (magenta) terminating on a cortical vessel in a healthy adult rat brain section (left) and in the peri-infarct region of the stroke hemisphere in a rat exposed to middle cerebral artery occlusion (model of stroke). Blue = DAPI; green = rat endothelial cell marker 1 (RECA-1).

Glial cells called astrocytes are abundant in the central nervous system. Increasingly, scientists are recognizing important contributions of astrocytes to many aspects of CNS function, one of them being neurovascular coupling – the process by which active neurons signal to blood vessels to increase the local blood flow and hence the supply of energy substrates. This process underlies several non-invasive neuroimaging techniques applied to human cognitive research and clinical diagnosis, such as functional magnetic resonance imaging. Under healthy mature conditions, neurovascular coupling is very robust, thus allowing this proxy measure to reliably report neuronal activity. However, the neurovascular coupling relationship is altered in pathological/disease contexts, which not only results in a mismatch of energy supply and demand in the brain, but also complicates the interpretation of neuroimaging data. Work in the Mishra lab is aimed at understanding the mechanisms of neurovascular coupling impairment in disease, particularly at the microvascular capillary level, with a focus on the role of astrocytes.

Neurovascular coupling impairment following stroke

A significant attenuation of neurovascular coupling is observed clinically in stroke patients for many years after the stroke. In the Mishra lab, we have revealed a similar suppression of neurovascular coupling at capillaries after experimental stroke models. This impairment occurs in peri-infarct tissue that otherwise looks healthy but displays changes in astrocyte morphology and expression patterns suggesting reactive astrogliosis. The functional outcomes manifested by these astrocytic changes are not understood. Current work in Dr. Mishra’s lab investigates the possibility that the constriction of capillaries and loss of neurovascular coupling observed after stroke may be due to pathophysiological signaling from reactive astrocytes. Some of the characteristics of these reactive astrocytes are reminiscent of developing astrocytes; thus, we are also studying how the neurovascular coupling response matures with the hope that this may help us understand how it is dysregulated after stroke.

Chronic effects of stroke-induced neurovascular impairment – role in dementia

Astrogliosis and neurovascular impairment are common features of many diseases of the central nervous system (including Alzheimer's disease, traumatic brain injury, vascular dementia, and chronic hypertension). Especially noteworthy are the epidemiological observations that patients harboring ischemic injuries develop dementia at a higher rate, and conversely, patients with dementia show evidence of ischemic damage in their brains. Thus, we are also investigating the long-lasting effects of mild strokes on neurovascular coupling and their contributions to cognitive loss.

Functions of astrocytic gap junctions

In a more basic research direction, we are also interested in how gap junctions help astrocytes do what they do. Although it has been known for many decades that astrocytes are coupled by gap junctions, the role of this coupling in neurovascular coupling, a prominent function of astrocytes, remains unknown. Thus, we are investigating whether gap junctional coupling of astrocytes contributes to their functions in K+ buffering and neurovascular coupling.

Members of the Mishra Lab smiling, standing in front of the Jungers Center for Neurosciences Research
Dr. Anusha Mishra, posed by a microscope

Anusha Mishra, Ph.D.
Principal Investigator

Anusha is interested in the physiological and pathological interactions between astrocytes and the cerebral microvasculature. Her interest in astrocytes began with the visualization of their fine ultrastructure while studying presynaptic plasticity using electron microscopy in Kristen Harris’s lab. She did her graduate work with Eric Newman at the University of Minnesota investigating astrocyte regulation of retinal vasculature in healthy and disease conditions. She then worked with David Attwell at University College London, where she demonstrated that astrocytes regulate capillary diameter in the cerebral cortex, and found evidence suggesting that ischemia-induced capillary constriction might underlie the no-reflow phenomenon following stroke. She joined OHSU in November 2016 and moved to the Jungers Center as an Assistant Professor in 2019. Her research focuses on astrocyte-dependent mechanisms of neurovascular coupling during development and following ischemic stroke.

Anusha also serves as a faculty member for the Neuroscience Graduate Program and the D3 hub of Graduate Program in Biomedical Sciences. She is also an active participant in the Department of Neurology DEI meetings.

Outside the lab, Anusha enjoys cooking, hiking, camping and reading fiction.

Headshot photo of Ozama "Oz" Ismail, Ph.D.

Ozama (Oz) Ismail, Ph.D.
Postdoctoral Scholar

Oz is a postdoctoral researcher studying the link between stroke and dementia. Within his project, he is interested in probing the lasting changes in cerebral blood flow and neurovascular coupling that follow ischemic events, and understanding how these changes contribute to the onset or acceleration of Alzheimer’s disease later in life.

Oz completed his PhD in neuroscience at University College London’s Centre for Advanced Biomedical Imaging. Prior to his PhD, he was the Centre’s Research Operations Manager and also worked as a Research Technician in collaboration with Eli Lilly & Company, developing imagining biomarkers for Alzheimer’s disease. During his PhD, he also did a short research placement in the Iliff Lab at OHSU, where he studied the molecular profiles of human Alzheimer’s disease subjects. Oz previously also worked at the Wellcome Trust Sanger Institute in Cambridge within the Mouse Genetics Programme. He studied physiology during his undergraduate degree at the University of Hertfordshire, with an industrial research placement in molecular biology at Brunel University in West London.

Away from the lab bench, Oz is an avid science communicator. He co-hosts a podcast called "Why Aren't You A Doctor Yet?" which tells compelling and diverse stories, combining science, tech and journalism with popular culture and comedy. He is also an advocate for diversity and co-founded the Minorities in STEM network in the UK to help support and showcase ethnic minorities in science. He currently sits on the board of the Alliance for Visible Diversity in Science at OHSU, serving as the Treasurer.

Headshot photo of Teresa Stackhouse

Teresa Stackhouse, B.A.
Graduate Student

Teresa received her B.A. in Biology from Lewis & Clark College in 2016, where she studied the role of phosphorylation on alpha-synuclein aggregation at the synapse in the lab of Dr. Tamily Weissman. Upon graduation, Teresa joined the Unni Lab at OHSU as a Research Assistant, where she continued studying mechanisms of alpha-synuclein aggregation until joining the Neuroscience Graduate Program at the Vollum Institute in 2018.

In the fall of 2019, Teresa joined the Mishra lab as a graduate student. Her major research interests include astrocyte biology and the role of glia in development and disease. Teresa is enthusiastic to combine these interests by studying common features of developing and reactive astrocytes and how this influences neurovascular coupling. Outside the lab, Teresa is passionate about broadening access to STEM education, particularly at the graduate and early career levels. She has been involved in Women in Science Portland and Alliance for Visible Diversity in Science at OHSU for a number of years.

Outdoors photo of Danica Bojovic

Danica Bojovic, B.A.
Graduate Student

Danica got her B.A. from Grinnell College, where she studied Biology and French. In the spirit of liberal arts education, she did several smaller research projects in neuroscience, psychology, and movement sciences. In neurosciences, she participated in two different projects, one studying the mechanism of homocysteine-induced exacerbation of oxidative stress in the mouse neuromuscular junction and the other studying brain atrophy in post-mortem brain tissues from Multiple Sclerosis patients. During her undergraduate studies, she became interested in glial cells and continued her graduate education in the Neuroscience Graduate Program at OHSU, studying astrocytes under a collaboration between the Mishra and von Gersdorff labs. Her thesis project focuses on the role of astrocyte gap junctions in potassium uptake and neurovascular coupling.

Outside of lab, Danica likes to cook, study languages, and socialize. She's interested in community building and promoting diversity in science. She is serving as Vice Chair of Administration at International Employee Resource Group (IERG) at OHSU.

Evan Calkins, in the lab

Evan Calkins, B.S.
Research Assistant/Lab Manager

Evan earned a B.S. in organismal biology from Portland State University, where he worked in the lab of Dr. Deborah Lutterschmidt studying the effect of environmental cues on the regulation and timing of garter snake behavior. During this time, he also performed a summer fellowship in the lab of Dr. Kari Buck in the Department of Behavioral Neuroscience at OHSU.

Following graduation, he worked for five years as a research assistant in the lab of Dr. Dennis Bourdette, where he investigated the protective and regenerative effects of thyromimetic compounds in demyelinating disorders (e.g., multiple sclerosis). He joined the Mishra lab in early 2020 and assists with several projects involving glial response and neurovascular coupling defects in stroke. He has also taken on an independent project analyzing the response of astrocytes and microglia to mild ischemic injuries. Outside of lab, Evan enjoys hiking, reading, and crossword puzzles.

Mechanisms of neurovascular coupling in development and disease

The brain requires high levels of oxygen and glucose for optimal function, but does not have significant metabolic substrate storage within its tissue. Increases in local neural activity trigger changes in cerebral blood flow to supply this increased energy demand. This process is termed neurovascular coupling is partly mediated by the release of vasoactive molecules from astrocytes in a Ca2+-dependent manner.

Three images and a graph describing the showing the response to U46619 and stimulation
Ex vivo neurovascular coupling assays. A cortical capillary showing constriction to U46619 (used as a pre-constrictor) and dilation to neuronal stimulation (3 s, 20 Hz). The trace on the left shows diameter change plotted over time, showing the response to U46619 and stimulation.

In healthy adult brains, neurovascular coupling leads to vascular dilation and increase in blood flow, which is reflected in human neuroimaging studies as an increase in blood oxygen level-dependent signals; however, in young rodents and humans, this response is often absent or negative. This early negative neurovascular coupling has important implications for brain development, but its mechanism is not understood.

Many neurological disorders are also associated with loss of neurovascular coupling, which can result in chronic metabolic deprivation and contribute to further neuronal dysfunction. In the case of ischemic stroke, loss or attenuation of neurovascular coupling is commonly observed in patients, and the Mishra lab has observed a similar loss in a rodent model of transient ischemic stroke. The mechanism for this loss of neurovascular coupling is also unknown.

We are using ex vivo neurovascular coupling assays and calcium imaging to study both these conditions to understand mechanisms regulating neurovascular coupling with a particular focus on the role of metabotropic glutamate receptor 5, which is expressed in young astrocytes and re-induced in reactive astrocytes.

Project lead: Teresa Stackhouse

Contribution of stroke-induced cerebrovascular impairments to cognitive dysfunction and dementia

As life expectancy of the global population increases, the incidence of Alzheimer’s disease and other dementias are beginning to climb significantly. Yet, there are still no effective cures or preventative treatments for the disease. Mounting research points towards a reduction in cerebral blood flow as one of the earliest changes that happen in the brains of individuals who go on to develop Alzheimer’s disease or other related forms of dementia.

One significant risk factor for dementia is a history of ischemic injuries. Patients who suffer strokes or transient ischemic attacks, or those who show evidence of silent strokes have a higher incidence of dementia. Conversely, a significant proportion of dementia patients show signs of silent or micro-infarcts, suggesting brief blockages of blood flow to regions of the brain. In this project, we aim to understand the long-term consequences of brief blockades of blood flow on brain regions outside the infarct that appear healthy after such a “silent” stroke.

TEMP ALT
Pseudocoloured perfusion map from a wild type adult mouse, computed using arterial-spin labeling magnetic resonance imaging (with corresponding color gradient scale: blue to red = low to high flow values). Image on the left depicts resting blood flow and image on the right depicts the increase in flow seen in the same brain following a hypercapnic challenge (by elevating the CO2 concentration in the breathing gas).

We utilize a variety of techniques, including magnetic resonance imaging to measure resting blood flow and vascular reactivity, laser Doppler flowmetry to quantify neurovascular coupling, behavioral assays to study cognition and immunohistological stains to assess pathophysiological burden. We have also applied the mild ischemia to a transgenic mouse model of amyloidosis to develop and investigate an innovative mixed-etiology dementia model. Findings from this project could shed light on how silent stroke triggers or accelerates the hallmarks of Alzheimer’s disease.

Project lead: Oz Ismail (collaboration with Dr. Martin Pike at the Advanced Imaging Research Center, OHSU and Dr. Laura Villasana at Legacy Research Institute; assisted by Evan Calkins)

Astrocyte gap junctions, potassium uptake, and neurovascular coupling

Astrocytes are coupled by gap junctions into a glial syncytium. Exactly how coupling between astrocytes helps them perform their critical functions remains unclear. In this project, we are interested in the contribution of astrocyte gap junctions in two key astrocyte functions: K+ buffering and neurovascular coupling. With their fine processes that concentrate around synapses and their endfeet that fully cover blood vessels in the brain, astrocytes are well poised to convey messages about neural activity to the vascular cells to induce neurovascular coupling.

Image showing an astrocyte patched with dye allowed to diffuse through the syncytium coupled by gap junctions. Several nearby astrocyte cell bodies and their endfeet on a nearby capillary are visible.
An astrocyte (*) was patched and dye allowed to diffuse through the syncytium coupled by gap junctions. Several nearby astrocyte cell bodies and their endfeet on a nearby capillary are visible.

At their peri-synaptic processes, astrocytes express neurotransmitters transporters as well as receptors and an abundance of the K+ channel Kir4.1. It is believed that astrocytes take up the K+ released during neuronal activity to prevent extracellular buildup and that the K+ can be buffered away to regions of low activity via gap junctions, thus maintaining neuronal extracellular homeostasis. However, evidence in support of this hypothesis is still murky. Furthermore, although it is known that K+ uptake can contribute to neurovascular coupling, whether gap junctional coupling of astrocytes modulates neurovascular coupling remains unknown. We are using ex vivo whole-cell patch clamping technique and vascular imaging to answer these questions.

Project lead: Danica Bojovic (co-mentored by Dr. Henrique von Gersdorff)

Selected publications

Li Z., McConnell HL, Stackhouse TL, Pike MM, Zhang W, Mishra A. 2021. Increased 20-HETE signaling suppresses neurovascular coupling after ischemic stroke in regions beyond the infarct. PRE-PRINT. bioRxiv.

Stackhouse TL and Mishra A. (2021) Neurovascular coupling in development and disease: focus on astrocytes. Frontiers in Cell and Development Biology. 9:702832. doi: 10.3389/fcell.2021.702832

ThorntonCA, MulqueenRM, TorkenczyKA, Nishida A, LowensteinEG, FieldsAJ, SteemersFJ, ZhangW, McConnellH, Woltjer RL, Mishra A, WrightKM and Adey AC. (2021) Spatially-mapped single-cell chromatin accessibility. Nature Communications 12, Article number: 1274.

Escartin C*, Galea E*, Lakatos A, O’Callaghan JP, Petzold GC, Serrano-Pozo A, Steinhauser C, Volterra A, Carmignoto G, Agarwal A, Allen NJ, Araque A, Barbeito L, Barzilai A, Bergles DE, Bonvento G, Butt AM, Chen WT, Cohen-Salmon M, Cunningham C, Deneen B, De Strooper B, Díaz-Castro B, Farina C, Freeman M, Gallo V, Goldman JE, Goldman SA, Götz M, Gutiérrez A, Haydon PG, Heiland DH, Hol EM, Holt MG, Iino M, Kastanenka KV, Kettenmann H, Khakh BS, Koizumi S, Lee CJ, Liddelow SA, MacVicar BA, Magistretti P, Messing A, Mishra A, Molofsky AV, Murai K, Norris CM, Okada S, Oliet SHR, Oliveira JF, Panatier A, Parpura V, Pekna M, Pekny M, Pellerin L, Perea G, Pérez-Nievas BG, Pfrieger FW, Poskanzer KA, Quintana FJ, Ransohoff RM, Riquelme-Perez M, Robel S, Rose CR, Rothstein J, Rouach N, Rowitch DH, Semyanov A, Sirko S, Sontheimer H, Swanson RA, Vitorica J, Wanner IB, Wood LB, Wu J, Zheng B, Zimmer ER, Zorec R, Sofroniew MV*, and Verkhratsky A*. (2021) Reactive astrocyte nomenclature, definitions, and future directions. Nature Neuroscience 24, 312–325.

*Howarth CH, *Mishra A. and *Hall CN. (2021) More than just summed neuronal activity: how multiple cell types shape the BOLD response. Philos Trans R Soc Lond B Biol Sci. 376(1815):20190630.

*McConnell HL, *Li Z, Woltjer RL and Mishra A. (2019) Astrogliosis and cerebrovascular dysfunction in neurological disorders: correlation or causation? Neurochemistry International; 128:70-84.

Nortley R, *Korte N, *Izquierdo P, *Hirunpattarasilp C, *Mishra A, *Jaunmuktane Z, *Kyrargyri V, Pfeiffer T, Khennouf L, Madry C, Gong H, Richard-Loendt A, Huang W, Saito T, Saido TC, Brandner S, Sethi H, Attwell D.  (2019) Amyloid beta oligomers constrict human capillaries in Alzheimer's disease via signalling to pericytes. Science; 365:eaav9518.

Methner C, Mishra A, Golgotiu K, Li Y, Wei W, Yanez ND, Zlokovic B, Wang RK, Alkayed NJ, Kaul S and Iliff JJ. (2019) Pericyte constriction underlies capillary de-recruitment during hyperemia in the setting of noncritical arterial stenosis. AJP Heart and Circulation Physiology; 317:H255-H263.

Mishra A. (2017) Binaural blood flow control by astrocytes: Listening to both synapses and the vasculature. Journal of Physiology; 595:1885-1902.

Mishra A, Reyno­­­­lds JP, Chen Y, Gourine AV, Rusakov DA and Attwell D. (2016) Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nature Neuroscience; 19:1619-27.

Attwell D, Mishra A, Hall CN, O’Farrell F and Dalkara T. (2015) What is a pericyte? Journal of Cerebral Blood Flow and Metabolism; 36:451-5.

*Hall CN, *Reynell C, *Gesslein B, *Hamilton NB, *Mishra A, Sutherland BA, O'Farrell FM, Buchan AM, Lauritzen M, Attwell D. (2014) Capillary pericytes regulate cerebral blood flow in health and disease. Nature; 508:55-60.

*Mishra A, *O’Farrell FM, Reynell C, Hamilton NB, Hall CN and Attwell D. (2014) ­­­Imaging pericytes and capillary diameter in brain slices and isolated retinae. Nature Protocols; 9:323-36.

Mishra A and Newman EA. (2012) Aminoguanidine reverses the loss of flicker-induced vasodilation in a rat model of diabetic retinopathy. Frontiers in Neuroenergetics.; 3:10.

Mishra A, Hamid AA and Newman EA. (2011) Oxygen modulation of neurovascular coupling in the rat retina. Proceedings of the National Academy of Sciences USA; 108:17827-31.

Mishra A and Newman EA. (2010) Inhibition of inducible nitric oxide synthase reverses the loss of functional hyperemia in diabetic retinopathy. Glia; 58:1996-2004.

A collage of members of the Mishra Lab working and living