Division of Glial Disease and Therapeutics

Astrocytic calcium signaling.

The last 20 years have witnessed a dramatic departure from the classical thinking: neurons are no longer believed to be the sole substrate of higher brain function and deciphering the role of neuroglia as active contributors to coordinated network activity has emerged as an exciting frontier in the study of neuroscience.

Interest in the study of neuroglia was greatly catalyzed in the 1990s when it was discovered that astrocytes –which throughout evolution increase in proportional makeup, cell size and complexity– respond to stimuli and the release of neurotransmitters via a special form of non-electrical excitability. In early work using cell cultures our group demonstrated that astrocytes respond to stimulation by fluctuations of the intracellular calcium ion (Ca²⁺) concentration, and that these fluctuations spread through networks of astrocytes physically coupled by gap-junctions. Although limited by the available experimental techniques, we observed that increases in astrocytic Ca²⁺ corresponded to changes in the synaptic activity of neighboring neurons. This finding, along with similar discoveries from independent labs led to an explosion of interest in the field of neuro-glial signaling that has since resulted in hundreds of publications implicating astrocytes as important contributors to what is now a shifting paradigm of information processing in the central nervous system (CNS).

Astrocytes surround neuronal synapses and form networks physically coupled by gap-junctions.

 Numerous follow-up studies have shown that activation of Ca²⁺ signaling in astrocytes can regulate both excitatory and inhibitory synaptic transmission, mediate essential physiological functions such as control over cerebral blood flow and respiratory rate, and influence state dependent changes in cortical activity during, for example, sleep and working memory. However, the exact mechanism for how astrocytes exert influence over neuronal networks remains a matter of intense controversy.

One popular model known as the tripartite synapse suggests that astrocytes, which extend processes that ensheathe neuronal synapses, detect the release of neurotransmitters and actively modulate pre- and post-synaptic neurotransmission by the calcium dependent release of gliotransmitters (i.e. transmitters released from glial cells that facilitate communication between neurons and other glia). However, recent evidence that astrocytes undergo developmental regulation of receptors sensitive to glutamatergic neurotransmission, and thus do not express receptors (e.g. mGluR5) thought to mediate gliotransmitter release in the adult brain, have questioned this model. Additionally, the functional significance of gliotransmission remains unclear due to the non-physiological nature of many of the experiments that gave rise to the concept.

What then might be the physiological function of astrocytic calcium signaling in the adult brain? Current work in our lab has suggested a simpler, but equally potent mechanism for astrocytic control of neuronal networks. In vivo imaging in the live rodent brain indicates that astrocytic calcium signaling corresponds with a decrease in extracellular potassium ion (K⁺) concentration, which in turn triggers neuronal hyperpolorization and suppression of excitatory synaptic activity. Although ion homeostasis has long been considered one of astrocyte’s passive housekeeping functions, our work suggests that Ca²⁺-dependent active uptake of K⁺ represents an active mechanism, capable of dynamically modulating the activity of neural circuits.

Further Reading

Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Sun W, McConnell E, Pare JF, Xu Q, Chen M, Peng W, Lovatt D, Han X, Smith Y, Nedergaard M. Science (New York, N.Y.). 2013 Jan 11; 339(6116):197-200.

Artifact versus reality--how astrocytes contribute to synaptic events. Nedergaard M, Verkhratsky A. Glia. 2012 Jul 0; 60(7):1013-23. Epub 2012 Jan 06.

Bergmann glia modulate cerebellar Purkinje cell bistability via Ca²⁺-dependent K⁺ uptake. Wang F, Xu Q, Wang W, Takano T, Nedergaard M. Proceedings of the National Academy of Sciences of the United States of America. 2012 May 15; 109(20):7911-6. Epub 2012 Apr 30.

Astrocytes modulate neural network activity by Ca²⁺-dependent uptake of extracellular K⁺. Wang F, Smith NA, Xu Q, Fujita T, Baba A, Matsuda T, Takano T, Bekar L, Nedergaard M. Sci Signal. 2012 Apr 3;5(218):ra26. doi: 10.1126/scisignal.2002334.

Extracellular Ca²⁺ acts as a mediator of communication from neurons to glia. Torres A, Wang F, Xu Q, Fujita T, Dobrowolski R, Willecke K, Takano T, Nedergaard M. Science signaling. 2012 Jan 24; 5(208):ra8. Epub 2012 Jan 24.

Glial calcium and diseases of the nervous system. Nedergaard M, Rodríguez JJ, Verkhratsky A. Cell calcium. 2010 Feb 0; 47(2):140-9. Epub 2009 Dec 31.

Astrocytic calcium signaling: mechanism and implications for functional brain imaging. Wang X, Takano T, Nedergaard M. Methods in molecular biology (Clifton, N.J.). 2009 489:93-109.

Astrocytic Ca²⁺ signaling evoked by sensory stimulation in vivo. Wang X, Lou N, Xu Q, Tian GF, Peng WG, Han X, Kang J, Takano T, Nedergaard M. Nature neuroscience. 2006 Jun 0; 9(6):816-23. Epub 2006 May 14.

New roles for astrocytes: redefining the functional architecture of the brain. Nedergaard M, Ransom B, Goldman SA. Trends in neurosciences. 2003 Oct 0; 26(10):523-30.

Intercellular calcium signaling mediated by point-source burst release of ATP. Arcuino G, Lin JH, Takano T, Liu C, Jiang L, Gao Q, Kang J, Nedergaard M. Proceedings of the National Academy of Sciences of the United States of America. 2002 Jul 23; 99(15):9840-5. Epub 2002 Jul 03.

ATP-mediated glia signaling. Cotrina ML, Lin JH, López-García JC, Naus CC, Nedergaard M. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2000 Apr 15; 20(8):2835-44.

ATP as a messenger in astrocyte-neuronal communication. Cotrina, M.; Nedergaard, M. The Neuroscientist. 2000; 6(2): 120-126.

Connexins regulate calcium signaling by controlling ATP release. Cotrina ML, Lin JH, Alves-Rodrigues A, Liu S, Li J, Azmi-Ghadimi H, Kang J, Naus CC, Nedergaard M. Proceedings of the National Academy of Sciences of the United States of America. 1998 95(26):15735-40.

Astrocyte-mediated potentiation of inhibitory synaptic transmission. Kang J, Jiang L, Goldman SA, Nedergaard M. Nature neuroscience. 1998 1(8):683-92.

Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Nedergaard M. Science (New York, N.Y.). 1994 Mar 25; 263(5154):1768-71.

Large (green) and small (red) tracers tagged to soluble proteins in the paravascular cerebrospinal fluid.

Throughout most of the body, a complex system of lymphatic vessels is responsible for cleansing the tissues of potentially harmful metabolic waste products, accumulations of soluble proteins and excess interstitial fluid. But astonishingly, the body’s most sensitive tissue –the central nervous system – lacks a lymphatic vasculature. What then accounts for the efficient waste clearance that must occur in order for the neural tissue of our brains to function properly?

This question has puzzled scientists for centuries. Our group believes that understanding how this process functions in the healthy nervous system holds the key to developing treatment options for a wide variety of neurological diseases, especially those characterized by the improper accumulation of misfolded proteins. The breakdown of the brain’s innate clearance system may in fact underlie the pathogenesis of neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s disease, in addition to ALS and chronic traumatic encephalopathy. Past efforts to explain how the brain cleanses parenchymal tissue have suggested that solute and fluid exchange occurs between the interstitial fluid and the cerebrospinal fluid, and that this exchange is driven by diffusion. Yet as many have noted, the distances for diffusion in the brain are too great to explain the highly regulated interstitial environment.

Recently, our lab has developed a fundamentally new approach to probing how the highly sensitive tissue of the CNS maintains its delicate extracellular environment. Our team made its initial discovery by injecting fluorescent tracers into the brains of living mice, and then imaging the movement of those tracers, in real time, using two-photon microscopy. These techniques allowed us to visualize the path of different sized molecules as they traversed the cortical layers of the brain, in addition to quantifying the clearance rate. Following the tracers revealed a distinctive pathway: once injected into the subarachnoid cerebrospinal fluid (CSF), the tracers readily flowed into the brain along the outside of the penetrating blood vessels. Further investigation confirmed that small channels 'piggybacking' the blood vasculature allow the CSF to flow into the brain tissue along para-arterial spaces and exit via a para-venous route. However, a critical component was still missing - based on diffusion alone, it would take over 100 hours for large solutes to traverse the inter- arterial and venous tissue.

Astrocytes (green) with AQP4 expression on astrocytic end-feet (purple), surrounding cerebral vasculature (white).

The final puzzle piece came in the form of astrocytes, or more specifically, long projections they extend that envelop the brain vasculature. These 'end-feet' densely express the water channel aquaporin-4, which allow astrocytes to act as intervening 'couplers' of a bulk interstitial fluid (ISF) flow. Transgenic animals that lacked aquaporin-4 exhibited a 70 percent reduction in the clearance of large solutes, such as Amyloid-β peptide, whose extracellular plaques are the hallmark of Alzheimer’s dementia. This 'peri-vascular' route for CSF-ISF exchange constitutes a complete anatomical pathway, which we dubbed the glymphatic system due to its dependence on glial cells performing a 'lymphatic' cleansing of the brain interstitial fluid.

Further characterization of the involvement of the glymphatic system in a number of neurological diseases and neurodegenerative disorders is underway. As an important further step to verify that the glymphatic system is functional in human brains, we are working in collaboration with researchers at the Stonybrook University to use contrast magnetic resonance imaging (MRI) to map regions of high and low volume solute exchange. Given that MRI is a commonly used clinical modality, it is our hope that this data may potentially enable the assessment of neurodegenerative disease risk and progression.

This project is supported by funding from NIH – National Institutes of Neurological Disorders and Stroke. (The Glymphatic System, a New Concept in Glia Biology).

Further Reading

Evaluating glymphatic pathway function utilizing clinically relevant intrathecal infusion of CSF tracer. Yang L, Kress BT, Weber HJ, Thiyagarajan M, Wang B, Deane R, Benveniste H, Iliff JJ, Nedergaard M. J Transl Med. 2013 May 1;11(1):107.

Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. Iliff JJ, Lee H, Yu M, Feng T, Logan J, Nedergaard M, Benveniste H. The Journal of Clinical Investigation. 2013;123:1299-1309.

A Paravascular Pathway Facilitates CSF Flow Through the Brain Parenchyma and the Clearance of Interstitial Solutes, Including Amyloid β. Iliff JJ, Wang M, Liao Y, Plogg BA, Peng W, Gundersen GA, Benveniste H, Vates GE, Deane R, Goldman SA, Nagelhus EA, Nedergaard M. Science Translational Medicine. 2012;147:1-11.

Human vs Rodent astrocytes. (Courtesy Alexi Verkhratsky (Chapter 3), Neuroglia by Kettenmann).

One of the most basic but fundamental questions underlying the study of brain science is understanding what factors account for the differences in interspecies intelligence and computational power. Sophisticated cognitive abilities, such as an unparalleled capacity for learning, language, abstract expression, and metacognition are said to set humans apart from animals. But what accounts for these differences? Classically, interspecies variation in intelligence has been attributed to neurons, the electrically excitable subunit of all nervous systems. However comparisons between the brains of different species indicate that the proportional make up and sophistication of a different, historically underappreciated, type of brain cell increases as a function of increased cognitive capability.

In species with more capable brains, astrocytes – a type of non-electrically excitable brain cell, also known as neuroglia— account for a considerably higher proportion of the brain. More importantly, human astrocytes are 20-fold larger and much more complex than rodent astrocytes. The correlation between proportional makeup and intelligence suggests that astrocytes, which were first discovered by Rudolf Virchow and later Camillo Golgi, in the late 1800’s, may be more involved in sophisticated neural processes than previously considered. Indeed, rapid advances in the field of glial biology have depicted a much more active role for astrocytes, one which is now widely considered to be critical for coordinating many basic physiological and higher order cognitive processes, such as control of respiratory rate and the dynamics of cerebral blood flow, regulation of the sleep wake cycle, and the facilitation of learning and memory, among others. Still remaining to be discovered are how human astrocytes functionally differ from their rodent counterparts and how these differences contribute to more complex information processing.

Mouse vs. human astrocytes.

 Nevertheless, observations of neuro-glial interactions have spurred great interest in developing glial targeted treatments for neurological disease. Although a number of glial targeted therapies, such as the treatment of neuroinflammation and chronic pain, have been shown to be effective on animal models in the laboratory setting, they have largely failed to predict human outcomes. The exact source of this discrepancy remains a contentious issue in science; however, recent research suggests that part of the reason may be due to differences in how astrocytes evolved in different species.

Efforts at contrasting the evolution of astrocytic form and function are ongoing, however, given that current neuroscience research and the development of treatments for neurological disease heavily depend on drawing parallels between humans and animal models, understanding the evolution and interspecies variability of astrocytes is particularly important. Work in our lab has demonstrated that compared to rodents, the average protoplasmic astrocyte from the human neocortex exhibits 10-fold more primary processes and is more than 20-fold larger in volume. Interestingly, our analysis using two-photon imaging of the photolysis of caged Ca²⁺ in fine astrocytic projections revealed that the speed at which astrocytes communicate with each other, via the propagation of calcium waves, is also faster in humans than it is in rodents.

Neurofilament staining of human astrocytes.

Our hope is that furthering our understanding of the evolution of astrocytes and how they differ between species will help overcome the obstacle of how to extrapolate and apply the findings from animal studies to human disease. Current work is focused on characterizing a fundamentally new way to study human glial cell interactions. We recently generated chimeric mice that have been engrafted with human glial progenitor cells that differentiated into human astrocytes. In an unexpected finding, as the engrafted human cells developed, they displaced the native mouse glial cells. Interestingly, the chimeric mice also exhibited heightened functions compared to control animals, such as improved learning and memory. As the first of its kind, this new model offers the unique opportunity to study the functions of human glial cells in animal models of neuropathologies, in vivo, in real time. Our hope is this work will further our understanding of the differences between rodent and human glial cells, and that it may lead to new insights into why animal models of disease often fail to predict human outcomes.

This project is a collaboration between the Goldman Lab and the Nedergaard Lab.

Further Reading

Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice. Han X, Chen M, Wang F, Windrem M, Wang S, Shanz S, Xu Q, Oberheim NA, Bekar L, Betstadt S, Silva AJ, Takano T, Goldman SA, Nedergaard M. Cell Stem Cell. 2013 Mar 7;12, 342–353.

Heterogeneity of Astrocytic Form and Function. Oberheim NA, Goldman SA, Nedergaard M. Methods Mol Biol. 2012;814:23-45. doi: 10.1007/978-1-61779-452-0_3. Review. PMID: 22144298.

Uniquely hominid Features of Adult Human Astrocytes. Oberheim NA, Takano T, Han X, He W, Lin JH, Wang F, Xu Q, Wyatt JD, Pilcher W, Ojemann JG, Ransom BR, Goldman SA, Nedergaard M. J Neurosci. 2009 Mar 11;29(10):3276-87. doi: 10.1523/JNEUROSCI.4707-08.2009. PMID: 19279265.

Astrocytic Complexity Distinguishes the Human Brain. Oberheim NA, Wang X, Goldman S, Nedergaard M. Trends Neurosci. 2006 Oct;29(10):547-53. Epub 2006 Aug 30. PMID: 16938356

Calcium signaling at the glial-vascular junction.

Thinking, reading, writing, throwing a baseball, or playing a musical instrument – are all actions that require spatially localized regions in the brain to undergo heightened neural activity. But when neurons undergo periods of intense activity, they demand oxygen and a replenishment of their depleted metabolic reserves. The question of how the brain knows exactly which regions to supply extra blood and nutrients to, a concept termed functional hyperemia, is one that underlies the very basis of human cognition and consciousness. Yet, despite decades of extensive study and its ubiquitous relevance to our moment-to-moment experience, it is a question that we are just now beginning to shed light on.

Although many of the brain’s higher order cognitive functions remain shrouded in mystery, recent advances in nuclear imaging modalities, such as MRI and PET have brought us closer to understanding the link between brain activity and simple behaviors. Blood-oxygen-level-dependent functional-MRI (BOLD fMRI) uses our understanding of hyperemia to functionally compartmentalize sensory and motor tasks within their anatomically discrete brain regions. The level of saturated deoxyhemogloben in blood produces an identifiable magnetic signal shift that can be contrasted to show which brain regions are demanding more blood; acting as a proxy for intense neural activity. However, despite its frequent use and powerful ability to map functional status, clinicians and researchers alike have only begun to understand the sequence of events that BOLD fMRI is actually imaging.

Our interest in hyperemia stems from the fact that despite being a hallmark of the healthy brain, the sequence of events and cellular players that orchestrate such a fundamental aspect of neurophysiology remain unknown. In studying functional hyperemia, we first recognized that neurons do not spatially interface with blood vessels. Heightened neural activity must therefore be acting through an intermediate to orchestrate changes in blood flow. Our early work demonstrated that astrocytes, which interface with neuronal synapses and project processes that surround the blood vasculature, react to neural activity in a Ca²⁺-dependent manner to signal localized changes in microcirculation. Using in vivo two-photon microscopy, we showed that photolysis of intracellularly-bound astrocytic calcium ions (in the astrocytic endfeet that surround blood vessels), resulted in relaxation of vascular smooth muscle cells and pericytes, to increase capillary flow. The vasodilation occurred in less than half a second following Ca²⁺ increase in the astrocytic endfoot and was dependent on the amount of released Ca²⁺.

Photolysis of caged calcium in the astrocytic end-foot.

Although it is now widely accepted that astrocytes exhibit control over local changes in cerebral blood flow, this model may not yet be complete. For example, other glial cells, such as oligodendrocytes and microglia may also play a role in coupling neural activity to blood flow. In addition to testing the involvement of various other cell types, further efforts are underway to characterize the hyperemic response following the activation of complex neural networks, with the hope of gaining insight into how coordination of astrocytic calcium signaling may facilitate higher order processes.

Further Reading

Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions. Kasischke KA, Lambert EM, Panepento B, Sun A, Gelbard HA, Burgess RW, Foster TH, Nedergaard M. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism. 2011 Jan 0; 31(1):68-81. Epub 2010 Sep 22.

Glial regulation of the cerebral microvasculature. Iadecola C, Nedergaard M. Nature neuroscience. 2007 Nov 0; 10(11):1369-76.

Two-photon imaging of astrocytic Ca2+ signaling and the microvasculature in experimental mice models of Alzheimer's disease. Takano T, Han X, Deane R, Zlokovic B, Nedergaard M. Annals of the New York Academy of Sciences. 2007 Feb 0; 1097:40-50.

Astrocyte-mediated control of cerebral blood flow. Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X, Nedergaard M. Nature neuroscience. 2006 Feb 0; 9(2):260-7. Epub 2005 Dec 25.

Signaling at the gliovascular interface. Simard M, Arcuino G, Takano T, Liu QS, Nedergaard M. Nature neuroscience. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2003 Oct 8; 23(27):9254-62.

Frank insult to the central nervous system (CNS), in the form of traumatic brain injury (TBI) or cerebral infarction (stroke), exposes the brain and spinal cord’s distinct cellular populations to an environmental catastrophe. Cell membranes and blood vessels rupture, spilling cytotoxic and inflammatory agents into the extracellular space and disrupting the blood supply, while blood brain barrier breakdown leads the influx of unfiltered blood born constituents, shutting down regular cellular metabolism. Such injuries result in highly variable functional outcomes depending on their location and severity and often result in intractable physical and emotional suffering, cognitive decline in the case of brain injuries, and the onset of dementia. Remarkably, despite the fact that most neuronal cell populations do not regenerate, trauma to the CNS is often followed by restoration of disrupted sensory and motor function. But on a cellular level, how does the body’s most sensitive tissue respond to trauma and what factors facilitate post-injury neuroplasticity?

Ex vivo image of diffuse microinfarction induced by carotid artery infusion of cholesterol crystals. GFAP (Blue) indicates reactive astrocytes, CD68 (Red) indicates activated microglia.

Key issues in translational neuroscience are a lack of understanding in the role that glial cells play in normal physiology and by extension the consequences of astrocytic dysfunction in pathophysiological conditions. Because complex cognitive and executive functions heavily depend on the brain’s interconnected neural network, localized injuries to one region often produce profound global effects on function. Our interest in the role of astrocytes following CNS injuries stems from the fact that they constitute therapeutic targets that occupy prime real-estate: by extending processes that interface with blood vessels and neuronal synapses, as well as forming physically coupled networks with other astrocytes, they effectively couple nutritional support with information transfer and processing. Moreover, emerging evidence suggests that the role of astrocytes is more active and dynamic than previously considered. Astrocytes have been shown to exert control over directing cerebral blood flow (hyperemia), mediate the clearance of pathologically relevant proteins (glymphatic system), and engage in sophisticated neuro-glial signaling interactions.

Mild vs. moderate rodent model of Hit and Run closed skull TBI, showing the time course of reactive gliosis. more...

 Despite being highly prevalent the pathogenesis of CNS injuries remains poorly understood. Experiments in our lab and others have suggested regardless of the source, acute CNS injuries evoke a similar cellular response: astrocytes and other glial cells such as microglia become activated in a process known as reactive gliosis. Although this phenomenon— first described almost a hundred years ago— is ubiquitous to a number of neuropathologies and has been extensively studied, the exact function of gliosis (whether it’s pathological or protective), its mechanisms of initiation, and its temporal relevance to therapeutic interventions following CSN injury remain controversial.

Work in our lab along with similar findings from independent labs have shown that in response to tissue trauma or non-physiological changes in the extracellular environment, astrocytes enlarge, proliferate in number, release long range signaling molecules such as ATP, and upregulate the expression intermediate filament proteins (e.g. GFAP) that are important for modifying cellular structure and astrocytic networks. Further complicating the picture of gliosis are results from experiments on animal models of traumatic brain injury, spinal cord injury and stroke, which have reported conflicting results as to its nature. These studies suggest that activation of astrocytes represents both a protective response aimed at ameliorating and stopping the spread of damage from the primary injury, as well as a pathological process that initiates secondary injury and profound disruptions to healthy function.

AQP4 de-polarization in reactive astrocytes after Hit and Run closed skull TBI.

Although the exact role of gliosis is not yet known, the development of animal models of injury and disease have aided tremendously in characterizing and interpreting the pathological significance of glial responses to CNS injuries. Routinely, however, successful treatments performed on animals fail to predict human outcomes. One of the major challenges is a lack of models that accurately reproduce relevant clinical features of CNS pathologies. For example, many animal models of TBI—the leading cause of injury related death in the United States—rely on fixation of the head and opening of the skull to facilitate direct cortical insult, as well as large doses of anesthesia. By and large, these injuries fail to reproduce one of the least understood components of common TBI pathologies, i.e. contracoup injuries, which result in damage both to the hemisphere of impact and the contralateral side.

In response to these shortcomings, we developed a novel Hit and Run model of closed skull traumatic brain injury that reproduces the clinical features of TBI, including contracoup injuries. Interestingly, we found that moderate and severe contusions to the intact rodent skull resulted in widespread astrogliosis (on both the ipsi- and contra- lateral hemispheres) and a significant disturbance in the typically polarized expression pattern of the astrocytic water channel aquaporin-4 (AQP4).

Schematic and 3 day representative image of diffuse lacunar infarction model in rodents. Reactive astrocytes are indicated by GFAP (blue), activated microglia by CD68 (red).

Similarly lacking are models for diffuse stroke, a pathology that is marked by cognitive decline and is thought to affect as large as 30% of the broader aged population. To gain a better understanding, we developed a model where we infused cholesterol crystals to induce multiple mini-strokes (diffuse lacunar infarctions) in rodents. In a surprising finding, we noted that unlike a typical macro-infarct where neuronal death in the affected tissue is largely complete within hours, micro-infarcts triggered delayed neuronal death that progressed gradually from ˜35% at 3 days to ˜60% at 28 days. Similar to our findings from ‘Hit and Run’ TBI, this time course coincided with widespread astrogliosis and corresponding global disruptions in AQP4 polarization. Such findings suggest that injury-specific responses of astrocytes may be the key to understanding how a localized tissue insult can lead to chronic cell death and global dysfunction.

Consequently, it is not surprising that astrocytes play important roles in the pathogenesis of various disease states including the primary and secondary stages of traumatic injury or cerebral infarct. Current work focuses on identifying therapeutic windows for altering the degree of injury-induced astrogliosis, as well as evaluating the functional consequences of the breakdown of pathologically relevant solute clearance via the glymphatic pathway.

This project is supported by funding from NIH – National Institutes of Neurological Disorders and Stroke. (Failure of Metabolite Clearance in a Model of Multi-Lacunar Infarcts).

Further Reading

'Hit & Run' model of closed-skull traumatic brain injury (TBI) reveals complex patterns of post-traumatic AQP4 dysregulation. Ren Z, Iliff JJ, Yang L, Yang J, Chen X, Chen MJ, Giese RN, Wang B, Shi X, Nedergaard M. J Cereb Blood Flow Metab. 2013 Jun;33(6):834-45.

Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts. Wang M, Iliff JJ, Liao Y, Chen MJ, Shinseki MS, Venkataraman A, Cheung J, Wang W, Nedergaard M. J Neurosci. 2012 Dec 12;32(50):17948-60.

Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury. Chen MJ, Kress B, Han X, Moll K, Peng W, Ji RR, Nedergaard M. Glia. 2012 Nov;60(11):1660-70.

Critical role of connexin 43 in secondary expansion of traumatic spinal cord injury. Huang C, Han X, Li X, Lam E, Peng W, Lou N, Torres A, Yang M, Garre JM, Tian GF, Bennett MV, Nedergaard M, Takano T. J Neurosci. 2012 Mar 7;32(10):3333-8.

Pericyte constriction after stroke: the jury is still out. Vates GE, Takano T, Zlokovic B, Nedergaard M. Nat Med. 2010 Sep;16(9):959.

A central role of connexin 43 in hypoxic preconditioning. Lin JH, Lou N, Kang N, Takano T, Hu F, Han X, Xu Q, Lovatt D, Torres A, Willecke K, Yang J, Kang J, Nedergaard M. J Neurosci. 2008 Jan 16;28(3):681-95.

Cortical spreading depression causes and coincides with tissue hypoxia. Takano T, Tian GF, Peng W, Lou N, Lovatt D, Hansen AJ, Kasischke KA, Nedergaard M. Nat Neurosci. 2007 Jun;10(6):754-62.

Angiogenic inhibition reduces germinal matrix hemorrhage. Ballabh P, Xu H, Hu F, Braun A, Smith K, Rivera A, Lou N, Ungvari Z, Goldman SA, Csiszar A, Nedergaard M. Nat Med. 2007 Apr;13(4):477-85.

Axial cross section of rodent spinal cord following acute contusion injury. Depicted in the center is the glial scar, consisting of an inner core of fibroblasts (blue) surrounded by a wall of reactive astrocytes (white).

Traumatic spinal cord injury (SCI) is a devastating assault to the central nervous system (CNS) that often results in permanent neurologic impairment, intense personal suffering and a disruption to essentially every aspect of life. An overwhelming majority of patients that suffer spinal cord injuries also develop chronic neuropathic pain syndromes that often persist indefinitely. Yet, despite high prevalence and decades of intensive study, to date, damage sustained from spinal cord injury is largely irreversible.

Pathologically, SCI manifests in two phases: the acute events surrounding primary injury typically result in damage or disturbance to the neuronal elements of the spinal cord, leading to the severing of axon tracts and neuronal cell death; however, within 1-3 days of the initial injury, SCI patients often experience a cascade of secondary pathological changes that amplify the initial traumatic injury and result in edema, spreading necrosis, inflammation, ischemia, and proliferation of reactive glial cells. Although little is known about the pathogenesis of this secondary phase, histological study has revealed a distinctive pattern of cellular events: in what appears to be a defense mechanism aimed at halting the spread of cellular damage, the injured neural tissue becomes isolated from surrounding healthy tissue by the formation of what is commonly referred to as the glial scar, consisting of a dense core of fibroblasts, surrounded by a symmetrical wall of reactive astrocytes.

Spinal cord injury induced Glial scar.

Following SCI, astrocytes – the largest and most abundant glial cell in the CNS – undergo reactive changes and form a barrier of projections linked together by gap-junctions. The hallmark of reactive changes in astrocytes, also known as “astrogliosis”, is the upregulation of the expression of intermediate filament proteins (e.g. glial-fibrillary-acid protein), and gap junction proteins (e.g. connexin 43) that together facilitate network communication and the passage of small intracellular molecules from one astrocyte to the next.

Further Reading

Critical role of connexin 43 in secondary expansion of traumatic spinal cord injury. Huang C, Han X, Li X, Lam E, Peng W, Lou N, Torres A, Yang M, Garre JM, Tian GF, Bennett MV, Nedergaard M, Takano T. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2012 Mar 7; 32(10):3333-8.

Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury. Chen MJ, Kress B, Han X, Moll K, Peng W, Ji RR, Nedergaard M. Glia. 2012 Nov 0; 60(11):1660-70. Epub 2012 Aug 01.

Connexin and pannexin hemichannels in inflammatory responses of glia and neurons. Bennett MV, Garré JM, Orellana JA, Bukauskas FF, Nedergaard M, Sáez JC. Brain research. 2012 1487:3-15. Epub 2012 Sep 10.

Functions of astrocytes and their potential as therapeutic targets. Kimelberg HK, Nedergaard M. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics. 2010 Oct 0; 7(4):338-53.

Physiological and pathological functions of P2X7 receptor in the spinal cord. Cotrina ML, Nedergaard M. Purinergic signalling. 2009 Jun 0; 5(2):223-32. Epub 2009 Feb 11.

Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, Takano T, Tian GF, Goldman SA, Nedergaard M. Proceedings of the National Academy of Sciences of the United States of America. 2009 Jul 28; 106(30):12489-93. Epub 2009 Jul 27.

Connexin 43 hemichannels are permeable to ATP. Kang J, Kang N, Lovatt D, Torres A, Zhao Z, Lin J, Nedergaard M. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2008 Apr 30; 28(18):4702-11.

P2X7 receptor inhibition improves recovery after spinal cord injury. Wang X, Arcuino G, Takano T, Lin J, Peng WG, Wan P, Li P, Xu Q, Liu QS, Goldman SA, Nedergaard M. Nature medicine. 2004 Aug 0; 10(8):821-7. Epub 2004 Jul 18.

Adenosine reduces transmission of painful input through pain-specific nerve fibers.

Chronic pain disrupts the lives of over one and a half billion people around the world, upsetting their sleeping habits, limiting their functional capabilities and causing severe emotional trauma. Though there is no effective long-term solution, we currently treat chronic pain patients with medications such as opioids (morphine, codeine, etc.), non-steroidals (ibuprofen, asprin, etc.) and certain anti-depressants and anti-epileptics (amitriptyline, duloxetine, gabapentin, pregabalin, etc.), which attenuate the sensory perception of acute pain, typically by disrupting or dampening the conduction of pain signaling along non-specific pathways. However, none of the current medications effectively treat chronic pain and many are accompanied by unacceptable side-effects, such as slowed cognition, gastrointestinal complications, and in certain cases of prolonged use, a paradoxical hyper-algesic effect.

Part of the problem may be that current treatment paradigms are based on a fundamentally incomplete understanding of chronic pain’s neurobiological basis. Historically, the medical literature has been primarily dominated by a neuronally-driven model of chronic pain, whereby pain-specific neurons become hyper-excitable and prone to spontaneous and ectopic activity. But this model is incomplete – what causes neurons to fire ‘spontaneously’, and what keeps them in an excitable state? Our lab investigates chronic pain from a totally different approach. We believe that broadening the pathological model to include glial-neuronal interactions will help answer those questions, and may well prove useful for developing cell-specific therapeutic targets.

We began our work by investigating the signaling interactions between astrocytes, and the pain-specific (alpha-delta and C-fiber) neurons of the spinothalmic tract. Astrocytes are prime suspects for chronic pain involvement, due to their widespread coupling via gap-junctions and close spatial proximity to neuronal synapses. In cell cultures, we demonstrated that astrocytes release ATP through unopposed connexin-43, a gap-junction protein typically coupled to other cells. Using bioluminscence in vivo imaging, we showed that wild type, but not mice lacking connexin-43 released large amount of ATP in a spinal cord injury model of chronic pain. Consequently, mice with deletion of connexin-43 developed less severe chronic central neuropathic pain after spinal cord injury. ATP is an evolutionarily conserved signal transmitter. Bacterial lysis releases ATP, which acts as a danger signal to surrounding bacterium, signaling them to disperse. It is therefore not surprising that release of ATP has evolved to play a role in pain signaling, the danger alert system of more complex eukaryotes.

The consequences of ATP release are significant, given that many cell types (including neurons and microglia) express purinergic (ATP) receptors. Astrocytic release of ATP may constitute a direct, upstream, signaling pathway, by which astrocytes contribute to the development and maintenance of chronic pain. ATP binding to purinergic receptors on microglia is known to trigger the release of cytokines and other pro-inflammatory and pro-nociceptive signaling molecules. The identification of an astrocyte-pain-specific neuron signaling pathway fundamentally changes the pathological model of chronic pain, and may hold the key to developing targeted therapies capable of disrupting the vicious cycle of pathological pain signaling. Although efforts are just beginning, we have already shown that purinergic receptor antagonists, such as Brilliant Blue G, are capable of disrupting the purinergic signaling pathway.

This project is supported by funding from NIH – National Institute on Drug Abuse. (Hemichannels, Astrocytic Release, and Neuropathic Pain).

Further Reading

Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury. Chen MJ, Kress B, Han X, Moll K, Peng W, Ji RR, Nedergaard M. Glia. 2012 Nov 0; 60(11):1660-70. Epub 2012 Aug 01.

Traditional acupuncture triggers a local increase in adenosine in human subjects. Takano T, Chen X, Luo F, Fujita T, Ren Z, Goldman N, Zhao Y, Markman JD, Nedergaard M. The journal of pain: official journal of the American Pain Society. 2012 13(12):1215-23.

Physiological and pathological functions of P2X7 receptor in the spinal cord. Cotrina ML, Nedergaard M. Purinergic signalling. 2009 Jun 0; 5(2):223-32. Epub 2009 Feb 11.

Deciphering migraine. Takano T, Nedergaard M. The Journal of clinical investigation. 2009 Jan 0; 119(1):16-9.

A needle a day. Inserting needles into certain acupuncture points, shown here as labeled dots, seems to spur tissue to release a pain-killing chemical. (Image courtesy of Dr. Takahiro Takano).

Since its inception in China around 2,000 B.C., acupuncture has been a popular modality of pain treatment in Eastern medical practices. The practice has since spread around the world and is commonly utilized as a therapeutic alternative to Western medicine. Traditional acupuncturists maintain that acupuncture’s analgesic effects come from manipulating the body’s natural energy or Qi, which is said to lie along meridians and become perturbed in the setting of pain or disease. Inserting fine needles into the tissue above important hubs of energy flow (acupoints), is thought to restore harmony between the opposing forces of Yin and Yang.

The US National Institutes of Health currently recognize that acupuncture can be a potent therapeutic alternative to conventional treatments for chronic pain. However, acupuncture’s grounding in metaphysical rather than biological justifications has made it a difficult target for empirical study. Our philosophy is that deciphering the biological basis for acupuncture represents a unique opportunity to study the neurobiology of chronic pain. An opportunity that not only holds the promise of improving acupunctures efficacy toward alleviating acute pain, but also of developing safe new alternatives to more effectively treat and manage chronic pain.

ATP released from fibroblasts after needle stimulation.

We first showed that tissue injury that is associated with the mechanical insertion and rotation of the acupuncture needle results in the release of the energy metabolite ATP. We tested the hypothesis that when the extracellular concentration of ATP exceeds the capacity for rapid removal, it will be enzymatically degraded and adenosine will accumulate, resulting in suppression of conductance of painful input, by activation of adenosine A1 receptors on peripheral pain fibers.

Using a mouse model of chronic neuropathic and inflammatory pain, we next showed that acupuncture reduces pain in wild type mice, but not in mice lacking adenosine A1 receptors. In another experiment, the injection of A1 receptor agonists into the Zusanli acupoint reduced the severity of pain, similar to the effect from acupuncture, in wild type but not in A1 receptor knockout mice. These experiments showed that needle manipulation is not necessary to reproduce acupunctures analgesic effect and highlighted the A1 receptor as a potential therapeutic target. In a subsequent study, we replicated our mouse findings in human volunteers, by showing that when an expert Chinese acupuncturist manipulates the Zusanli acupoint, there is a similarly robust elevation in extracellular adenosine.

Schematic of acupuncture induced analgesic pathway.

Collectively our work amounts to a first peak into the mechanistic underpinnings of acupuncture, the formal study of which is actually in its infancy, despite having a rich history and worldwide practice. Ongoing work focuses on how the elevation of adenosine levels in the periphery correlate to responses in the central nervous system, as well as ways by which the analgesic effect of acupuncture can be pharmacologically enhanced or replicated independent of needle manipulations.

Further Reading

Fibroblast cytoskeletal remodeling induced by tissue stretch involves ATP signaling. Langevin HM, Fujita T, Bouffard NA, Takano T, Koptiuch C, Badger GJ, Nedergaard M. J Cell Physiol. 2013 Sep;228(9):1922-6.

Cellular control of connective tissue matrix tension. Langevin HM, Nedergaard M, Howe AK. 2013 Aug;114(8):1714-9.

Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts. Goldman N, Chandler-Militello D, Langevin HM, Nedergaard M, Takano T. Cell Calcium. 2013 Apr;53(4):297-301

Traditional acupuncture triggers a local increase in adenosine in human subjects. Takano T, Chen X, Luo F, Fujita T, Ren Z, Goldman N, Zhao Y, Markman JD, Nedergaard M. The journal of pain: official journal of the American Pain Society. 2012 13(12):1215-23.

Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture. Goldman N, Chen M, Fujita T, Xu Q, Peng W, Liu W, Jensen TK, Pei Y, Wang F, Han X, Chen JF, Schnermann J, Takano T, Bekar L, Tieu K, Nedergaard M. Nature neuroscience. 2010 Jul 0; 13(7):883-8. Epub 2010 May 30.