Advances in Bioscience and Biotechnology, 2013, 4, 73-80 ABB http://dx.doi.org/10.4236/abb.2013.48A2010 Published Online August 2013 (http://www.scirp.org/journal/abb/) Pro-inflammatory cytokine; tumor-necrosis factor-alpha (TNF-α) inhibits astrocytic support of neuronal survival and neurites outgrowth Ebtesam M. Abd-El-Basset Department of Anatomy, Faculty of Medicine, Kuwait University, Kuwait City, Kuwait Email: [email protected] Received 16 June 2013; revised 16 July 2013; accepted 31 July 2013 Copyright © 2013 Ebtesam M. Abd-El-Basset. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT 1. INTRODUCTION Reactive astrogliosis has been implicated in the fail- Astrocytes support the CNS neurons, by controlling the ure of axonal regeneration in adult mammalian Cen- microenvironment surrounding the neurons, picking up tral Nervous System (CNS). It is our hypothesis that any excess neurotransmitters or ions that are secreted inflammatory cytokines act upon astrocytes to alter from the neurons during conduction of nerve impulses their biochemical and physical properties, which may and metabolic activity [1,2]. Astrocytes are also impor- in turn be responsible for the failure of neuronal re- tant in secreting several trophic factors, vital to the survi- generation. We have therefore examined the effect of val of neurons [3,4]. Astrocytes can promote neurite out- tumor-necrosis factor-alpha (TNF-α) on the ability of growth both in vitro and in vivo. They express receptors astrocytes to support the survival of the cortical neu- for a variety of growth factors and neurotansmittors [5]. Nerve growth factors production in astrocytes is regula- rons and the growth of the neurites. Mouse astrocytes ted by various cytokines including TNF-α [6]. In addi- and cortical neuronal cultures were prepared. It was tion, astrocytes play a crucial part in the antioxidant de- observed that when neurons were cultured in absence fence of the brain [7]. of astrocytes only a few of them grew and survived However, in cases of insult to the CNS, astrocytes only for 5 - 6 days. These neurons had small cell bo- begin to accumulate at the margin of the injury forming a dies and few, short neurites. However, when the same layer that interfaces between the injury and intact tissues, numbers of neurons were cultured on the top of as- which become known as the glial scar or reactive astrog- trocytes, more neurons grew and survived up to 16 - liosis [8]. Reactive astrogliosis is characterized by an 18 days. They had bigger cell bodies and many long increase in the amount of GFAP which is regulated by branched neurites that formed anestamosing net- hormones, cytokines, and growth factors [9,10]. Al- works. The neurons then coalesced and the neurites though the functional role of the glial scar is not yet formed thick bundles. When the same numbers of completely understood, it has been suggested that the neurons were grown on the top of astrocytes pre- scar inhibits nerve regeneration by acting as a physical treated with TNF-α, few neurons survived up to 13 barrier that neurons cannot penetrate [8,11]. However, days. The neurites of the survived neurons were the formation of the glial scar can also be beneficial in shorter than neurites of neurons grown on normal isolating the damaged region and in preventing the astrocytes and did not form bundles. In addition, spread of the inflammatory response to intact neuronal TNF-α stimulated the expression of glial fibrillary tissue [12,13] thereby helping to limit tissue degenera- acidic protein (GFAP) by astrocytes. These results tion and preserving function after CNS injury. In addi- support that the pro-inflammatory cytokine, TNF-α tion, recent data showed axon regeneration through scars modulates the gliosis and that the astrocytic cell sup- and into sites of chronic spinal cord injury [14]. All these ports neuronal survival and neurite outgrowth. data emphasize that reactive astrocytes play a role in wound healing and restoring CNS function at the site of Keywords: Astrocytes; Neurons; Cytokines; injury. The data also indicate the importance of pre- Tumor-Necrosis Factor-Alpha serving astrocyte populations [13,15,16]. OPEN ACCESS 74 E. M. Abd-El-Basset / Advances in Bioscience and Biotechnology 4 (2013) 73-80 During injury to the CNS, astrocytes are also respon- Guidelines for Care and use of laboratory Animals and sible for the secretion of cytokines, such as interleukin-1 the Guide for the Care and Use of Laboratory animals (IL-1), interleukin-6 (IL-6) and TNF-α [17]. Cytokines (Kuwait University-Faculty of Health Publication). All are responsible for certain changes that take place in the efforts were made to minimize animal suffering, to re- nervous system [18]. It is known that the immune system duce the number of animals used, and to utilize alter- and the nervous system communicate with each other natives to in vivo techniques. through the sharing of biochemical informational sub- stances and their receptors, which is referred to as neu- 2.1. Astrocyte Cultures ral-immune interactions. For instance, several neurotran- The astrocyte cultures were prepared according to the smitters, neuroendocrine hormones and even nerve method of [24]. The cerebral hemispheres of newborn growth factors are produced by immune cells and clearly Balb/c mice were isolated aseptically and the meninges play an important role in the nervous system [4,6]. The were removed. The neopallia was dissected out and then CNS has its own resident immune system in which glia forced through a sterile 75 µm Nitex mesh. The cells cells (astrocytes and microglia) not only serve supportive were suspended in a modified Eagle’s Minimum Essen- and nutritive roles for neurons but also engage from time tial Medium (mMEM) containing 5% horse serum. For to time in several inflammatory processes that defend the culturing, 5 × 104 nigrosine-excluding cells were plated CNS from pathogens and help it to recover from stress in 60 mm Petri dishes containing 22 × 22 mm glass co- and injury. These normal glia functions are kept by nor- verslips in a total volume of 3 ml growth medium. Ali- mal activity of neurons [19]. The immune status of the quots of 5 × 106 nigrosine-excluding cells were plated in CNS is strictly regulated in healthy brain in which the 75 cm2 culture flasks. The cultures were incubated at immune response is kept to a minimum by activity of 37˚C in a humidified atmosphere of 5% CO2 in air. After neurons. Loss of neurons or their physiological activity 3 days of incubation the growth medium was removed, would render the CNS recognizable by invading immune the cell debris and non-attached cells were washed off cells and can sometimes result in a more severe neuro- and fresh medium was added and was changed every 2 - inflammatory cycle that promotes neurodegenerative di- 3 days. After 10 days some culture plates were incubated seases [20]. for 5 days and culture flasks for 2 and 8 days in medium TNF-α, a potent proinflammatory molecule, is mainly to which 200 international units (U)/ml of TNF-α (Sigma, produced by astrocytes in the CNS. During brain injury St. Louis, MO) had been added. The new medium was or inflammatory processes TNF-α is produced by acti- also changed every 2 - 3 days. vated microglia, astrocytes and macrophages [17,21]. In culture, astrocytes secret TNF at low level but the secre- 2.2. Neuronal Cultures tion is increased after treatment with inflammatory sti- muli such as interferon-gamma combined with IL-1beta. The cerebral neuronal cultures were prepared according TNF-α promotes the proliferation of astroglia and mi- to the method of [25]. The outer parts of the brain of croglia and therefore may be involved in pathological embryonic mice foetuses (E 15) were used. The cells of processes such as astrogliosis and demyelination [22]. the brains were separated in 0.25% trypsin in Puck’s TNF-α triggers downstream signaling cascades that con- solution for 5 min. at room temperature. Horse serum trol a number of cellular processes related to cell viabi- was added to stop the trypsin action, and the tissue was lity, gene expression, ion homostasis and synaptic inte- triturated by pipetting up and down. The suspensions grity [23]. It should be indicated that there is limited were centrifuged at 1000 rpm for 5 min. After removal of information available on the effect of TNF-α on astro- supernatant, the cells were resuspended in mMEM con- cytes-cortical neurons interactions. It is our hypothesis taining 30 mili-moles (mM) glucose and filtered through that inflammatory cytokines act upon astrocytes to alter a sterile 75 µm Nitex mesh. For culturing 5 × 104 nigro- their biochemical and physical properties, which may in sine-excluding cells were plated in 60 mm Petri dishes turn be responsible for the failure of neuronal regenera- containing 22 × 22 mm glass coverslips coated with tion. We have therefore examined the effect of TNF-α on poly-L-lysine (Sigma) in a total volume of 3 ml growth the ability of astrocytes to support the survival of the medium. After incubation in serum-free medium for 15 neurons and the growth of the neurites in mixed neuron- min. at 37˚C, the culture was rinsed once with fresh astrocyte cultures. The latter provides a valuable in vitro medium and incubated in freshly prepared medium model system for studying the direct interactions be- containing 5% hourse serum and 30 mili-moles (mM) tween neurons and astrocytes. glucose, with changes of medium every 2 - 3 days.