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This work describes the development of electroconductive hydrogels as injectable matrices for neural tissue regeneration by exploiting a biocompatible conductive polymer - poly(3,4-ethylenedioxythiophene)- poly(styrenesulfonate) (PEDOT:PSS) - combined with a biomimetic polymer network made of gelatin. Our approach involved also genipin - a natural cross-linking agent - to promote gelation of gelatin networks embedding PEDOT:PSS. The achieved results suggest that physical-chemical properties of the resulting hydrogels, like impedance, gelation time, mechanical properties, swelling and degradation in physiological conditions, can be finely tuned by the amount of PEDOT:PSS and genipin used in the formulation. Furthermore, the presence of PEDOT:PSS (i) enhances the electrical conductivity, (ii) improves the shear modulus of the resulting hydrogels though (iii) partially impairing their resistance to shear deformation, (iv) reduces gelation time and (v) reduces their swelling ability in physiological medium. Additionally, the resulting electroconductive hydrogels demonstrate enhanced adhesion and growth of primary rat cortical astrocytes. Given the permissive interaction of hydrogels with primary astrocytes, the presented biomimetic, electroconductive and injectable hydrogels display potential applications as minimally invasive systems for neurological therapies and damaged brain tissue repair.

reserved 10 si Fund for Investment in Basic Research (FIRB)–Futuro in Ricerca (RBFR12SJA8_001; RBFR12SJA8_002) This work was supported by grants from the University of Bologna (RFO2016/17; to M.C.) and the Italian Minister for Education, University, and Research within the FIRB–Futuro in Ricerca (to V.B. and G.P.N.). The U.S. Air Force Office of Scientific Research, Research Projects Advanced Nano-Structured Material Interfaces and Devices for InVivo-like–In Vitro Monitoring of Astrocytes Physiology and Brain Toxicology (ASTROMAT; FA9550 16 1 0502) and Shedding Light on Glial Function (ASTRONIR; FA9550-17-1-0502) (both to V.B.) Consolidated evidence indicates that astroglial cells are critical in the homeostatic regulation of cellular volume by means of ion channels and aquaporin-4. Volume-regulated anion channel (VRAC) is the chloride channel that is activated upon cell swelling and critically contributes to cell volume regulation in astrocytes. The molecular identity of VRAC has been recently defined, revealing that it belongs to the leucine-rich repeat-containing 8 (LRRC8) protein family. However, there is a lack of evidence demonstrating that LRRC8A underpins VRAC currents in astrocyte. Nonetheless, direct evidence of the role of LRRC8A in astrocytic regulatory volume decrease remains to be proved. Here, we aim to bridge this gap in knowledge by combining RNA interference specific for LRRC8A with patch-clamp analyses and a water-permeability assay. We demonstrated that LRRC8A molecular expression is essential for swelling-activated chloride current via VRAC in primary-cultured cortical astrocytes. The knockdown of LRRC8A with a specific short interference RNA abolished the recovery of the cell volume after swelling induced by hypotonic challenge. In addition, immunoblotting, immunofluorescence, confocal imaging, and immunogold electron microscopy demonstrated that LRRC8A is expressed in the plasma membrane of primary cortical astrocytes and in situ in astrocytes at the perivascular interface with endothelial cells. Collectively, our results suggest that LRRC8A is an essential subunit of VRAC and a key factor for astroglial volume homeostasis mixed Formaggio F, Saracino E, Mola MG, Rao SB, Amiry-Moghaddam M, Muccini M, Zamboni R, Nicchia GP, Caprini M, Benfenati V. Formaggio F, Saracino E, Mola MG, Rao SB, Amiry-Moghaddam M, Muccini M, Zamboni R, Nicchia GP, Caprini M, Benfenati V.

The physiological ability of the mammalian CNS to integrate peripheral stimuli and to convey information to the body is tightly regulated by its capacity to preserve the ion composition and volume of the perineuronal milieu. It is well known that astroglial syncytium plays a crucial role in such process by controlling the homeostasis of ions and water through the selective transmembrane movement of inorganic and organic molecules and the equilibration of osmotic gradients. Astrocytes, in fact, by contacting neurons and cells lining the fluid-filled compartments, are in a strategic position to fulfill this role. They are endowed with ion and water channel proteins that are localized in specific plasma membrane domains facing diverse liquid spaces. Recent data in rodents have demonstrated that the precise dynamics of the astroglia-mediated homeostatic regulation of the CNS is dependent on the interactions between water channels and ion channels, and their anchoring with proteins that allow the formation of macromolecular complexes in specific cellular domains. Interplay can occur with or without direct molecular interactions suggesting the existence of different regulatory mechanisms. The importance of molecular and functional interactions is pinpointed by the numerous observations that as consequence of pathological insults leading to the derangement of ion and volume homeostasis the cell surface expression and/or polarized localization of these proteins is perturbed. Here, we critically discuss the experimental evidence concerning: (1) molecular and functional interplay of aquaporin 4, the major aquaporin protein in astroglial cells, with potassium and gap-junctional channels that are involved in extracellular potassium buffering. (2) the interactions of aquaporin 4 with chloride and calcium channels regulating cell volume homeostasis. The relevance of the crosstalk between water channels and ion channels in the pathogenesis of astroglia-related acute and chronic diseases of the CNS is also briefly discussed.

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult.

Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca 2+ and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca 2+ response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brain's volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.

Astroglial cell survival and ion channel activity are relevant molecular targets for the mechanistic study of neural cell interactions with biomaterials and/or electronic interfaces. Astrogliosis is the most typical reaction to in vivo brain implants and needs to be avoided by developing biomaterials that preserve astroglial cell physiological function. This cellular phenomenon is characterized by a proliferative state and altered expression of astroglial potassium (K(+)) channels. Silk is a natural polymer with potential for new biomedical applications due to its ability to support in vitro growth and differentiation of many cell types. We report on silk interactions with cultured neocortical astroglial cells. Astrocytes survival is similar when plated on silk-coated glass and on poly-D-lysine (PDL), a well known polyionic substrate used to promote astroglial cell adhesion to glass surfaces. Comparative analyses of whole-cell patch-clamp experiments reveal that silk- and PDL-coated cells display depolarized resting membrane potentials (-40 mV), very high input resistance, and low specific conductance, with values similar to those of undifferentiated glial cells. Analysis of K(+) channel conductance reveals that silk-astrocytes express large outwardly delayed rectifying K(+) current (K(DR)). The magnitude of KDR in PDL- and silk-coated astrocytes is similar, indicating that silk does not alter the resting K(+) current. We also demonstrate that guanosine- (GUO) embedded silk enables the direct modulation of astroglial K(+) conductance in vitro. Astrocytes plated on GUO-embedded silk are more hyperpolarized and express inward rectifying K(+) conductance (K(ir)). The K(+) inward current increases and this is paralleled by upregulation and membrane polarization of K(ir)4.1 protein signal. Collectively these results indicate that silk is a suitable biomaterial platform for the in vitro studies of astroglial ion channel responses and related physiology. (c) 2010 Elsevier Ltd. Al! rights reserved.