Search

Introduction - Gastro-intestinal nematodes are important helminth parasites in all animal species. However, they must regarded particularly dangerous in domestic ruminant species, also in relation to consequent economic losses. Aim - The present study focused on providing current data, missing since several decades, on gastro-intestinal nematode parasitic infection, prevalence and epidemiology in adult cattle (dairy and brood cows) bred in Italy. Materials and methods - The survey was performed collecting 427 fecal samples from a bovine slaughterhouse in the province of Bologna (Italy). Samples, obtained from single animals processed, were analyzed by qualitative coprological examinations. From the same animals 100 abomasa were randomly selected and examined by necropsy technique to assess the presence of worm burdens. Results - Gastro-intestinal nematode eggs were detected in 31% of individual fecal samples examined. Evaluation of abomasa exhibited a prevalence of 13% of helminthes. Ostertagia, Trichostrongylus and Cooperia were the isolated genera. The fecal output of nematode eggs was significantly related with the livestock category and the stocking density. Discussion - The influence of livestock category on the occurrence of positive coprological results can be attributed to the condition of animal husbandry: brood cows are often pasture raised. The correlation observed between positive samples and herd size, with intermediate class (50-99 animals) associated with higher prevalence, may be explained by a different effectiveness of hygiene management among classes of stocking density. Conclusions - The study results show that endoparasitic infection by nematodes is a problem that must be considered ubiquitous in Italy in adult cattle, with relatively high prevalence rate. Nevertheless, it seems to be still underestimated by technicians in the field.

The basic principle of biomimicry is currently adopted in tissue engineering approach for the design of the majority of the three-dimensional (3D) scaffolds, both in terms of physicochemical properties, as well as bioactivity. Custom-made scaffold fabrication methodologies can be successfully used to manipulate biocompatible materials (synthetic and natural ones), thus assuring an improved performance of the scaffolds through an accurate design of matrix pores and degradation properties to fully match the specific requirements of natural tissue to be regenerated. However, extensive studies have highlighted some intrinsic limitation of synthetic polymers in terms of biological performance, thus requiring the need of tailored functionalization via bioactive molecules (i.e., proteins, growth factors). Hence, proteins of natural origin may represent the most attractive alternative to synthetic polymers, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as their excellent biocompatibility. This chapter aims at classifying the most interesting natural proteins suitable for the fabrication of three-dimensional porous scaffolds, by remarking main advantages in different biologically and clinically relevant applications.

Astroglial ion channels and calcium signaling play a central role in the physiology and pathophysiology of the Central Nervous System. In this context, increasing efforts are needed to generate innovative tools for monitoring astrocytes biochemical or bioelectrical activity in vitro and in vivo. Organic field effect devices have a great potential for generating advanced biomedical tools to enable real-time recording and manipulation of communication signals between neural cells. 1,2,3 We previously reported on transparent Organic Cell Stimulating and Sensing Transistors (O-CSTs) that provide bidirectional stimulation and recording of primary neurons.1,2 The transparency of the device also allows the optical imaging of the modulation of the astroglial Ca2+ signaling bioelectrical activity.4,5 Here we explore O-CST functionality to stimulate, evoke and control astroglial Ca2+ signaling and whole cell conductance in primary cultured astrocytes. We found that primary astroglial cells can adhere, grow and differentiate on the perylene based field-effect transistor. Furthermore, the organic material preserves astrocytes electrophysiological properties. By means of patch-clamp analyses, we explore the effect of the stimulation on the whole-cell conductance of patched astrocytes. We found that the stimulation lead to an exclusive increase in the inward current that could be prevented by the application of Ruthenium Red and RN-1734 prior to stimulation. This finding suggests a contribution of the transient receptor potential (TRP) channels, of which TRPV-4 has been shown in former studies to mediate Ca2+ influx in astrocytes. We show, that the provided O-CST evokes intracellular astrocytic Ca2+ response, which can be determined by calcium imaging. The evoked signal was blocked by Gadolininium(III)-chloride and Ruthenium red, thus underpinning the involvement of TRPV channels. By using Ca2+-free bath saline, we show that the response is due to an influx of external Ca2+. We also explored the cellular volume changes and cell viability after stimulation. Our organic cell stimulating and sensing transistor paves the way to a new generation of devices for stimulation, manipulation and recording of astroglial cells' bioelectrical activity in vitro. [1] Benfenati, V. et al. Nat. Mater. 12, 672-680 (2013) [2] Toffanin, S. et al. J. Mater. Chem. B 1 , 3850-3859 (2013) [3] Ichikawa, M. et al. US20130153884 A1, 17/08/2011 [4] Scemes, E. Giaume, C. Glia. 54, 716-25 (2006) [5]Gee, KR. et al. Cell Calcium. 27, 97-106 (2002) [6] Cold Spring Harb Protoc; doi 10.1101/pdb.top066050

There is a demand of technologies targeted to provide ad- vance in electrophysiological knowledge on neurons and astrocytes, that might help in understanding brain func- tion/disfunction. In this context, promising opportunities are offered by exploitation of combined functionalities of or- ganic semiconductors films such as softness, optical trans- parency, optoelctronic properties and iono/electronic con- ductivity, in innovative device platform. Recently, by whole- cell patch-clamp recording, extracellular recording, calcium imaging and confocal microscopy investigations, we provide consolidated evidence that, Organic-Cell stimulating and Sensing Transistor architecture (O-CST), based on perylene derivative organic semiconductor, is enabling bidirectional communication with primary neurons with high signal-to- noise ratio. O-CST operation can also stimulate sustained intracellular calcium signalling and whole-cell inward con- ductance in primary astrocytes. The biophysics and phar- macology of astroglial ion channels, underpinning the O-CST evoked response is provided. The perspective offered by com- bination of 2D/3D organic biofunctional interface with or- ganic bioelectronic and light emitting device is discussed un- derlining the potential of the emerging field of organic neuro- optoelectronics.

Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long-term effects on both neurones and astroglial cells. Whether Guo also modulates the activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 microm) promotes the functional expression of an inward rectifier K+ (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up-regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo-induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo-induced up-regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that down-regulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K+ homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K+-buffering impairment of the astroglial syncytium that occurs in pathological conditions.

Astroglial ion channels and aquaporins are key player in the maintenance of cerebral homeostasis. Alteration in their patchy distribution in the plasma-membrane of astrocytes is suggested to be involved in vivo in acute brain pathophysiologies (hypoxia/ischemia, edema). Currently available in vitro models hamper to study astroglial membrane channels in an in vivo-like expression pattern since their distribution is more mislocalized in the cytosol as well as in the whole plasma membrane. We sought to overcome this issue by using nanostructured materials, processed as film, named hydrotalcite nanoparticles (HTlc NP). Due to their chemical composition and ordered lamellar structure, HTlc enable hosting and controlled delivery of wide range of molecules (including peptides, siRNAs and oligonucleotides). Thus they have a huge potential as in vitro substrates to be used for interaction with neural cellular systems. Here, we evaluated the effect of the interaction of HTlc NP films on primary cultured rat neocortical astrocytes. Molecular and functional properties of astrocytes grown on HTlc films were compared to those of astrocytes grown on Poly-D-Lysine (PDL). Cell viability assay showed that HTlc-films supported astrocytes adhesion and survival with value comparable to those of PDL. Morfological analyses revealed that astrocytes grown on HTlc NP films displayed a starlike morphogy typical of differentiated astrocytes in vivo. Western blot (WB) analyses and immunofluorescence (IF) confocal imaging revealed that stellation was not accompanied by GFAP upregulation, indicating that differentiation was not due to occurrence of gliotic reaction. WB, IF and whole cell patch-clamp revealed that differentiation was accompanied by molecular and functional up-regulation and polarized membrane expression of both inward rectifying potassium channel Kir 4.1 and aquaporin 4, AQP4. Collectively, these results indicate that HTlc NP films are suitable substrates to generate novel in vitro models where to modulate, alter and study the specific functionality and expression of membrane channels of astrocytes. Sponsored by: Futuro in Ricerca (MIUR) RBFR12SJA8, EU-ITN-OLIMPIA GA 316832, Progetto Bandiera Fabbrica del Futuro Silk-it

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 basic principle of biomimicry is currently adopted in tissue engineering approach for the design of the majority of the three-dimensional (3D) scaffolds, both in terms of physicochemical properties, as well as bioactivity. Custom-made scaffold fabrication methodologies can be successfully used to manipulate biocompatible materials (synthetic and natural ones), thus assuring an improved performance of the scaffolds through an accurate design of matrix pores and degradation properties to fully match the specific requirements of natural tissue to be regenerated. However, extensive studies have highlighted some intrinsic limitation of synthetic polymers in terms of biological performance, thus requiring the need of tailored functionalization via bioactive molecules (i.e., proteins, growth factors). Hence, proteins of natural origin may represent the most attractive alternative to synthetic polymers, mainly due to their similarities with the extracellular matrix (ECM), chemical versatility as well as their excellent biocompatibility. This chapter aims at classifying the most interesting natural proteins suitable for the fabrication of three-dimensional porous scaffolds, by remarking main advantages in different biologically and clinically relevant applications.

Silk fibroin (SF) is a natural polymer of increasing interest for applications ranging from tissue engineering to optoelectronics. Here, we report a new mild and facile strategy targeted on hydroxylic pendants of serine and tyrosine residues, to functionalize SF in water, based on the use of amino(propyl)triethoxysilane (APTES), a common silylating agent. APTES is exploited as a bifunctional linker to bind SF through the triethoxysilane a-ends and to simultaneously graft species of interest, even hydrophobic ones, by means of the end g-amino groups. By using a fluorescent oligothiophene bearing amino reacting end groups (T3) we monitor the process simply through fluorescence detection and we demonstrate the value of the proposed method to achieve chemically modified SF materials. Moreover, we show that the new SF based biocomposite retains silk filmability and transparency but also shows T3 fluorescence and markedly enhanced mechanical robustness.

In this study, we investigated the influence of processing methods on the chemo-physical properties of silk fibroin (SF) film and on their interaction with neural cells. Structural, thermal and morphological analysis revealed a strong correlation between the conformation, stability and texture of silk films and the fabrication conditions. An increase in temperature, methanol treatment and the use of a microfluidic approach led to an improvement in SF film stability in terms of beta-sheet content, mechanical resistance, dissolution and enzymatic degradation. An effect on the interaction of SF films with neural cells, through a modulation of the surface properties, was also observed. In particular, hydrophobic surfaces induce proliferation of astrocytes and neuron adhesion whereas hydrophilic surfaces promote a remarkable neurite outgrowth. A detailed knowledge of the effect of manufacturing parameters on SF film properties can facilitate and extend the applications of silk-based biomaterials in tissue engineering and drug release systems.

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.

Prostate cancer (PC) represents the most common type of cancer among males and is the second leading cause of cancer death in men in Western society. Current options for PC therapy remain unsatisfactory, since they often produce uncomfortable long-term side effects, such as impotence (70%) and incontinence (5–20%) even in the first stages of the disease. Light-triggered therapies, such as photodynamic therapy, have the potential to provide important advances in the treatment of localized and partially metastasized prostate cancer. We have designed a novel molecular conjugate (DR2) constituted of a photosensitizer (pheophorbide a, Pba), connected to a nonsteroidal anti-androgen molecule through a small pegylated linker. This study aims at investigating whether DR2 represents a valuable approach for PC treatment based on light-induced production of single oxygen and nitric oxide (NO) in vitro. Besides being able to efficiently bind the androgen receptor (AR), the 2-trifluoromethylnitrobenzene ring on the DR2 backbone is able to release cytotoxic NO under the exclusive control of light, thus augmenting the general photodynamic effect. Although DR2 is similarly internalized in cells expressing different levels of androgen receptor, the AR ligand prevents its efflux through the ABCG2-pump. In vitro phototoxicity experiments demonstrated the ability of DR2 to kill cancer cells more efficiently than Pba, while no dark toxicity was observed. Overall, the presented approach is very promising for further development of AR-photosensitizer conjugates in the multimodal photodynamic treatment of prostate cancer.

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.