Introduction to Astroglia
Basic Cell Functions and Applications in Research

The human brain consists of approximately one trillion cells, of which approximately 10% are considered to be neurons, and the remaining 90% are support cells called glia.¹ One type, Astroglia, often called astrocytes, are one of the major types of glial cell.² The brains of smaller mammalian creatures, naturally, have a smaller number of cells; and the ratio of astrocytes to neurons declines.³

The term astrocyte derives from a combination of a Greek word for star (astron; plural, astra) and the scientific word for cell (cyte, which is in turn derived from the Greek word kytos, meaning vessel). Astrocytes are characteristic of star-shaped glial cells in the brain and spinal cord.

Astrocytes are classically identified using histological analysis; many of these cells express the intermediate filament glial fibrillary acidic protein (GFAP). Several forms of astrocytes exist in the central nervous system including fibrous (in white matter), protoplasmic (in grey matter), and radial astrocytes.

The fibrous glia cell is usually located within white matter, has relatively few organelles, and exhibits long unbranched cellular processes. This type often has "vascular feet" that physically connect the cells to the outside of capillary walls when they are in proximity to them. The protoplasmic glia cells are the most prevalent and are found in grey matter tissue. They possess larger quantities of organelles, and exhibit short and highly branched tertiary processes.

The radial glia cell is disposed in planes perpendicular to the axes of ventricles. One of their processes abuts the pia mater, while the other is deeply buried in gray matter. Radial glia cells are mostly present during development, playing a role in neuron migration. Müller cells of the retina and Bergmann glia cells of the cerebellar cortex represent an exception, being present still during adulthood. When in proximity to the pia mater, all three forms of astrocytes send out processes to form the pia-glial membrane.

Functions of Astrocytes

As shown in figures 1 and 2, Astrocytes perform many functions like, biochemical support of endothelial cells that form the blood–brain barrier, provision of nutrients to the nervous tissue, maintenance of extracellular ion balance, and have a role in the repair and scarring process of the brain and spinal cord following traumatic injuries.⁴

Figure 1

Figure 2

Some of the Key Functions of Astrocytes Are Described in More Detail Below
 

• Structural: Astrocytes are the most abundant glial cells in the brain that are closely associated with neuronal synapses. They regulate the transmission of electrical impulses within the brain.
   

• Glycogen Fuel Reserve Buffer: Astrocytes contain glycogen and are capable of glycogenesis. The astrocytes next to neurons in the frontal cortex and hippocampus store and release glycogen. Thus, astrocytes can fuel neurons with glucose during periods of high rate of glucose consumption and glucose shortage.
   

• Metabolic Support: Astrocytes provide neurons with nutrients such as lactate.
   
• Blood–Brain Barrier: The blood–brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions.
   

• Regulation of blood flow: Astrocytes make extensive contacts and have multiple bidirectional interactions with blood vessels, including regulation of local CNS blood flow. Recent findings show that astrocytes produce and release various molecular mediators, such prostaglandins (PGE), nitric oxide (NO), and arachidonic acid (AA), that can increase or decrease CNS blood vessel diameter and blood flow in a coordinated manner. Moreover, astrocytes may be primary mediators of changes in local CNS blood flow in response to changes in neuronal activity. Astrocytes have processes in contact with both blood vessels and synapses. Via these contacts, astrocytes titrate blood flow in relation to levels of synaptic activity.
   
• Transmitter uptake and release: Astrocytes express plasma membrane transporters such as glutamate transporters for uptake & release of several neurotransmitters, including glutamate, ATP, and GABA. More recently, astrocytes were shown to release glutamate or ATP in a vesicular, Ca2+-dependent manner.
   
• Regulation of ion concentration in the extracellular space: Astrocytes express potassium channels at a high density. When neurons are active, they release potassium, increasing the local extracellular concentration. Because astrocytes are highly permeable to potassium, they rapidly clear the excess accumulation in the extracellular space. If this function is interfered with, the extracellular concentration of potassium will rise, leading to neuronal depolarization. Abnormal accumulation of extracellular potassium is well known to result in epileptic neuronal activity.
   
• Modulation of synaptic transmission: In the supraoptic nucleus of the hypothalamus, rapid changes in astrocyte morphology have been shown to affect heterosynaptic transmission between neurons. In the hippocampus, astrocytes suppress synaptic transmission by releasing ATP, which is hydrolyzed by ectonucliotidases to yield adenosine. Adenosine acts on neuronal adenosine receptors to inhibit synaptic transmission.
   
• Promotion of the myelinating activity of oligodendrocytes: Electrical activity in neurons causes them to release ATP, which serves as an important stimulus for myelin to form. However, the ATP does not act directly on oligodendrocytes. Instead, it causes astrocytes to secrete cytokine leukemia inhibitory factor (LIF), a regulatory protein that promotes the myelinating activity of oligodendrocytes.
   
• Nervous system repair: Upon injury to nerve cells within the central nervous system, astrocytes fill up the space to form a glial scar, repairing the area by transformation into neurons

Astrocytes in Neuropathology

Astrocytes are fundamental for the control of brain homeostasis and they also represent an important part of the intrinsic brain defense system. Brain insults of multiple etiologies trigger an evolutionary conserved astroglial defense response generally referred to as reactive astrogliosis (Figure 3).

The astrogliosis is essential for both limiting the areas of damage (by scar formation through anisomorphic astrogliosis) and for the post-insult remodeling and recovery of neural function (by isomorphic astrogliosis). Astrocytes are involved in all types of brain pathologies from acute lesions (trauma or stroke) to chronic neurodegenerative processes such as Alexander’s disease, Alzheimer’s disease (Figure 4), Parkinson’s disease, multiple sclerosis and psychiatric diseases.

The pathologically relevant neuroglial processes are many, and they include various programmes of activation, which are essential for limiting the areas of damage, producing neuro-immune responses and for the post-insult remodeling and recovery of neural function. Recent studies also emphasized the role of astroglial degeneration and atrophy in the early stages of various neurodegenerative disorders, which may be important for cognitive impairments. All in all astroglial cells determine to a very large extent the progression and the outcome of neurological diseases.⁷

Applications of Astrocytes in Research

Astrocyte mediated Neurotoxicity Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system “All in all, astroglial cells determine to a very large extent the progression and the outcome of neurological diseases” may prove more relevant to human CNS structure and function than neuronal cells alone. High Content Analysis based assays by co-culture of neurons and astrocytes, enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and its development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.⁸

In Vitro Applications of Primary Astrocyte Cultures

The possibilities for studying astrocytes in a physiological setting have faced issues of resolution, distinction between cell types accounting for the observed effects and lack of sufficiently sophisticated in vivo techniques. Such issues have been overcome by the use of primary astrocyte cultures, where detailed studies can be carried out, e.g. employing electrophysiological, biochemical, molecular and genetic tools. Manipulation of cultured astrocytes such as the gene expression, knockdown of a specific gene with siRNA, where the roles of a single gene can be studied in detail, is a valuable tool to gain detailed knowledge of specific mechanisms in astrocytes. In addition, functional studies of receptors expressed by astrocytes and their responses can be studied in real time in astrocyte cultures, giving indications of their functional roles and identification of potent drug candidates for neurodegenerative disorders.⁹

Astrocyte-Neuron Co-culture Models

The different neural cell types living in close proximity in brain, modulate each other in numerous ways. A monotypic in vitro culture system does not provide a milieu where interactions and adaptations to the surrounding environment are required. A means of addressing the lack of the dynamic microenvironment in vitro, while still maintaining the advantages of monotypic astrocyte cultures is by setting up co-culture system. Astrocytes have been co-cultured with neurons on a confluent layer of astrocytes. Neuron and astrocyte co-culture studies help to examine cell surface molecule expression and trophic factor release. Alternatively, astrocytes can be cultured in plates, where inserts with neurons, oligodendrocytes, neuronal stem cells, microglia or endothelial cells are added, so that they share the same medium, but are grown in separate layers. A more dynamic in vivo-like environment is thus created for the cells, enabling the researcher to investigate cell-type specific effects separately.⁹

Astrocytes in Blood Brain Barrier Model

In vitro cell culture models have provided a great deal of information about the induction of the BBB phenotype in brain endothelium, and have generally confirmed the key inductive role of astrocytes. Freshly isolated brain endothelial cells and some immortalized brain endothelial cell lines will grow as a flat monolayer on plastic or on porous filter inserts, and will retain aspects of a BBB phenotype, but generally with some loss of barrier function. Some of these properties can be up-regulated by co-culture with cells of glial origin, such as primary astrocytes.¹⁰

Astrocytes in Three Dimensional Culture Models

3D cell culture techniques for culturing astrocytes & neurons provide highly useful tools for studying neurogenesis, neuropathology, toxicology and effects of new drug targets. They also allow the potential for side by side comparisons of disease state cells versus healthy controls for the study of neurological disorders and diseases. The use of such systems overcomes some of the limitations of standard 2D culture systems in which cells are cultured as a monolayer. The recapitulation of the complex microenvironment in which cells exists, allows 3D culture systems to bridge the gap between traditional cell culture approaches and in vivo models such as transgenic mice. By mimicking the physiological environments both mechanically and spatially, the crosstalk between the cells and environment leads to a closer scenario to what can be seen in the brain. There are number of different approaches to 3D culture of cells including generation of organoids/spheroids or scaffolding systems such as collagen hydrogels, RAFT^TM Technology and nanofiber scaffolds.

Astrocytes and In Vitro Model of Reactive Astrogliosis

Astrogliosis, whereby astrocytes in the central nervous system (CNS) become reactive in response to tissue damage, is a prominent process leading to the formation of the glial scar that inhibits axon regeneration after CNS injury. Upon becoming reactive, astrocytes undergo various molecular and morphological changes including upregulating their expression of GFAP and chondroitin sulfate proteoglycans (CSPGs) as well as other molecules that are inhibitory to axon growth. In an in vitro model of reactive astrogliosis, astrocytes treated with transforming growth factor-β (TGF-β), induces increased expression as well as secretion of CSPGs. These reactive astrocytes show inhibitory effects on neuron growth. These reactive astrocytes provide a vehicle for testing substances that might overcome the glial scar and promote regeneration.¹¹

Astrocytes in Cell Therapy

It deserves brief mention that transplantation strategies involving astrocytes are also under investigation. For example, grafts of stem cells/progenitor cells that mature into healthy astrocytes are reported to improve outcome in a mouse model of Amyotrophic Lateral Sclerosis (ALS), in which host astrocytes are abnormal and express a mutant Superoxide Dismutase (SOD). A different strategy uses grafts of astrocytes that are genetically modified to produce specific molecules, such as growth factors, as therapeutic pumps to deliver those molecules in specific locations. Such grafts of genetically modified astrocytes may be able to provide long-term, locally restricted delivery of therapeutic molecules via cells that integrate into the neural parenchyma both structurally and functionally.¹²

Astrocyte Gene Expression Profiling Studies

Future advances in understanding how astrocytes contribute to diseases of neurodevelopment will rely heavily on identification of biomarkers responsible for the disease. Once identified, relevant gene loci can be used to drive transgenes to confirm specificity to the astrocyte lineage. Many genes show dynamic expression during development and their key regulatory elements could be used to drive conditionally inducible genes affecting astrocyte function.¹³

Astrocyte related Research Tools at Lonza

Lonza provides high-quality cryopreserved astrocytes (mouse, rat & human origin), growth medium, and Nucleofector^TM Technology for supporting the key in vitro research applications of astrocytes. Lonza products related to Astrocyte research has been used by researchers worldwide and has been cited in high quality international scientific journals.^14-20

References

1. Oberheim NA, Wang X, Goldman S, Nedergaard M. Astrocytic complexity distinguishes the human brain. Trends in Neurosciences (2006); 29: 547–553

2. Kettenmann H, Ransom B. The concept of neuroglia: a historical perspective. In: Kettenmann H, Ransom BR, editors. Neuroglia. 2nd ed. New York: Oxford University Press (2005); 1–16

3. Nedergaard M, Ransom B, Goldman SA. New roles for astrocytes: redefining the functional architecture of the brain. Trends in Neurosciences (2003); 26: 523–530

4. Fiacco TA, Agulhon C, McCarthy KD. Sorting out Astrocyte Physiology from Pharmacology. Annual Review of Pharmacology & Toxicology (2009); 49 (1): 151–74

5. Sarma PotukuchiVenkata Gurunadha Krishna, Srikanth Lokanathan, Vengamma Bhuma, Venkatesh Katari, Chandrasekhar Chodimella, Mouleshwara Prasad BodapatiChandra, Sanjeevkumar Akula. In vitro differentiation of cultured human CD34+ cells into astrocytes. Neurology India (2013); 61 (4): 383–388

6. Figley CR & Stroman PW. The role(s) of astrocytes and astrocyte activity in neurometabolism, neurovascular coupling, and the production of functional neuroimaging signals. European Journal of Neuroscience (2011); 33: 4: 577–588

7. Michael V, Sofroniew, Harry V & Vinters. Astrocytes: biology and pathology. Acta Neuropatholgica (2010); 119: 7–35

8. Anderl JL, Redpath aS, Ball AJ. A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity. Journal of Visualized. Experiments (2009); 27: e1173

9. Sofie CL, Lasse KB, Helle SW, Arne S, and Michael DN. Primary cultures of astrocytes: Their value in understanding astrocytes in health and disease. Neurochemical Research (2012); 37; 11; 2569–2588

10. N. Joan Abbott. Astrocyte–endothelial interactions and blood–brain barrier permeability. Journal of Anatomy (2002); 200: 629–638

11. Panpan Yu, Hang Wang, Yasuhiro Katagiri, and Herbert MG. An in vitro model of reactive astrogliosis and its effect on neuronal growth. Methods in Molecular Biology (2012); 814: 327–340

12. Mary EH and Michael VS. Reactive astrocytes as therapeutic targets for CNS disorders. Neurotherapeutics (2010); 7: 494-506

13. Anna VM, Robert K, Erik U, Hui-hsin T, Benjamin D, William DR, Ben AB, and David HR. Astrocytes and disease: a neurodevelopmental perspective. Genes & Development (2009); 26: 891–907

14. Deborah C, Anvita K, Vyacheslav A, Fatah K, and Lena A. Human immunodeficiency virus-restricted replication in astrocytes and the ability of gamma interferon to modulate this restriction are regulated by a downstream effector of the wnt signaling pathway. Journal of Virology (2007); 6: 5864–5871

15. Frank JB, Juan Carlos RV, Nancy HM, Ofelia FA and Juan Pablo RV. RIG-I contributes to the innate immune response after cerebral ischemia. Journal of Inflammation (2015); 12: 52

16. Roger SC, Milena P, Justin D, Carolyn EK, Carole B, Johanne WA, Juan H, Javier C, Yee Kee JL, Adam KW, Samantha JF, Sarah AD, Melinda F, Lyn DB, Meng IC, James CV, and Adrian KW. Redefining the role of metallothionein within the injured brain. The Journal of Biological Chemistry (2008); 283: 22; 15349–15358

17. Fei X, Aya K, Hiroshi OMW, Sudha KP, Puspa RP, Shigeru H, Andrew W, Yin-Yuan Mo, Brian EM, Wen L, Koji F, Megumi I, Sambad S, Yin Liu, Kerui Wu, Elizabeth P,KW. Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain. EMBO Molecular Medicine (2013); 5; 384-396

18. Neha VS, Benjamin BG, Chaitanya J, Kathleen B, and Anuja G. Astrocyte elevated gene-1 is a novel modulator of HIV-1-associated neuroinflammation via regulation of nuclear factor-B signaling and excitatory amino acid transporter-2 repression. The Journal of Biological Chemistry (2014); 289; 28: 19599–19612

19. Masuhiro N, Kazuhisa D, Sanae K, Osamu K, Shinsaku N and Aiko Y. Pharmacological assessment of ARTCEREB irrigation and perfusion solution for cerebrospinal surgery using primary cultures of rat brain cells. The Journal of Toxicological Sciences, (2010); 35: 4: 447-457

20. Martin H, Anita MH, Andreas P, Dorothea SA, Anna-Maria O, Norbert K, Fausto M, Andreas F, Hans-Ulrich H, Harald Staiger. Insulin promotes glycogen storage and cell proliferation in primary human astrocytes. PLOS ONE (2011); 6: 6; e21594