3D Printed Customizable Microsampling Devices for Neuroscience Applications.

Multifunctional devices that incorporate chemical or physical measurements combined with ways to manipulate brain tissue via drug delivery, electrical stimulation, or light for optogenetics are desired by neuroscientists. The next generation in vivo brain devices will likely utilize the extensive flexibility and rapid processing of 3D printing. This Perspective demonstrates how close we are to this reality for advanced neuroscience measurements. 3D printing provides the opportunity to improve microsampling-based devices in ways that have not been previously available. Not only can 3D printing be used for actual device creation, but it can also allow printing of peripheral objects necessary to assemble functional devices. The most probable 3D printing set up for microsampling devices with appropriate nm to µm feature size will likely require 2-photon polymerization-based printers. This Perspective describes the advantages and challenges for 3D printing of microsampling devices as an initial step to meet the next generation device needs of neuroscientists.

Attachment and optimization of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms to a 3D printed lattice.

A lattice was designed and fabricated using three-dimensional (3D) printing that allows for the facile transfer of biofilms formed from either Staphylococcus aureus, Staphylococcus epidermidis, or Pseudomonas aeruginosa into a fresh cell culture flask. To enhance biofilm production onto the filaments, three protein-based treatments were compared: fetal bovine serum (FBS), bovine serum albumin (BSA), and fibrinogen (Fb). Protein treatments included either supplementing the growth broths or pre-coating the lattice prior to immersion into the broth. S. aureus and P. aeruginosa biofilms were observed on all tested filaments that contained the supplement Fb. S. epidermidis required BSA to form biofilm. Ultimately, polycarbonate (PC) was chosen as the optimal material for lattice creation since it can be autoclaved without warping key design features. In addition, this 3D printed design may facilitate biofilm transfer from the bacterial culture to different cell culture platforms.

Probing spatial inhomogeneity of cholinergic changes in cortical state in rat.

Acetylcholine (ACh) plays an essential role in cortical information processing. Cholinergic changes in cortical state can fundamentally change how the neurons encode sensory input and motor output. Traditionally, ACh distribution in cortex and associated changes in cortical state have been assumed to be spatially diffuse. However, recent studies demonstrate a more spatially inhomogeneous structure of cholinergic projections to cortex. Moreover, many experimental manipulations of ACh have been done at a single spatial location, which inevitably results in spatially non-uniform ACh distribution. Such non-uniform application of ACh across the spatial extent of a cortical microcircuit could have important impacts on how the firing of groups of neurons is coordinated, but this remains largely unknown. Here we describe a method for applying ACh at different spatial locations within a single cortical circuit and measuring the resulting differences in population neural activity. We use two microdialysis probes implanted at opposite ends of a microelectrode array in barrel cortex of anesthetized rats. As a demonstration of the method, we applied ACh or neostigmine in different spatial locations via the microdialysis probes while we concomitantly recorded neural activity at 32 locations with the microelectrode array. First, we show that cholinergic changes in cortical state can vary dramatically depending on where the ACh was applied. Second, we show that cholinergic changes in cortical state can vary dramatically depending on where the state-change is measured. These results suggests that previous work with single-site recordings or single-site ACh application should be interpreted with some caution, since the results could change for different spatial locations.

Microdialysis Sampling of Quorum Sensing Homoserine Lactones during Biofilm Formation.

Bacteria communicate chemically through a system called quorum sensing. In this work, microdialysis sampling procedures were optimized to collect quorum sensing molecules produced during in situ biofilm formation directly on the polymeric semipermeable membrane of the microdialysis probe. V. harveyi, a Gram-negative bacterium, was used as the model organism and releases variable chain length acylhomoserine lactones (AHLs) and acyl-oxohomoserine lactones (AOHLs) as signaling molecules during quorum sensing. Eliciting biofilm formation required coating fetal bovine serum onto the poly(ether sulfone) microdialysis membrane. Dialysates were collected in different experiments either during or after biofilm formation directly on a microdialysis probe. Continuous sampling of C4-AHL, C6-AHL, C8-AHL, C6-OXO-AHL, and C12-OXO-AHL was achieved over a period of up to 4 days. The AHLs and AOHLs in dialysates were concentrated with solid-phase extraction and quantified using LC-MS. Dialysate concentrations obtained for the AOHLs and AHLs ranged between 1 and 100 ppb (ng/mL) and varied between sampling days. This work demonstrates the initial use of microdialysis sampling to collect quorum sensing signaling chemicals during biofilm formation by a Gram-negative bacterial species.

In Situ Inner Lumen Attachment of Heparin to Poly(ether sulfone) Hollow Fiber Membranes Used for Microdialysis Sampling.

An in situ chemical surface modification method to attach heparin to the inner lumen of a single hollow fiber poly(ether sulfone) (PES) membrane incorporated into a commercial microdialysis sampling device is described. The immobilization process uses gentle, room-temperature conditions with the enzyme laccase (E.C. 1.10.3.2) and 4-hydroxybenzoic acid (4HBA). The resulting functionalized inner membrane surface with a carboxylic acid functional groups allowed for (1-ethyl-3-(3-(dimethylamino)propyl) carbodimide)/ N-hydroxysuccinimide) EDC/NHS chemistry to attach heparin to the membrane surface. X-ray photoelectron spectroscopy measurements suggested successful attachment of 4HBA polymers and heparin onto the PES membrane. The microdialysis extraction efficiency after membrane surface modification was measured with model compounds fluorescein isothiocyanate (FITC)-labeled dextrans and lysozyme and the cytokines acidic fibroblast growth factor (aFGF) and CXCL1 (KC/GRO). This work demonstrates an in situ method to modify commercially available PES hollow fiber microdialysis membranes with amine or carboxylic acid functional groups.

Iloprost Affects Macrophage Activation and CCL2 Concentrations in a Microdialysis Model in Rats.

PURPOSE: The hypothesis that locally-released iloprost, a synthetic prostacyclin analog, affects macrophage phenotype at a microdialysis implant in the subcutaneous space of rats was tested. Macrophage activation towards alternatively-activated phenotypes using pharmaceutical release is of interest to improve integration of implants and direct the foreign body reaction toward a successful outcome. METHODS: Macrophage cell culture was used to test iloprost macrophage activation in vitro. Microdialysis sampling probes were implanted into the subcutaneous space of Sprague-Dawley rats to locally deliver iloprost in awake- and freely-moving rats. Monocyte chemoattractant protein -1 (CCL2) was quantified from collected dialysates using ELISA. Immunohistochemical staining was used to determine the presence of CD163+ macrophages in explanted tissues. RESULTS: Iloprost reduced CCL2 concentrations in NR8383 macrophages stimulated with lipopolysaccharide. CCL2 concentrations in collected dialysates were similarly reduced in the presence of iloprost. Iloprost caused an increase in CD163+ cells in explanted tissue surrounding implanted microdialysis probes at two days post probe implantation. CONCLUSIONS: Localized delivery of iloprost caused macrophage activation at the tissue interface of a microdialysis subcutaneous implant in rat. This model system may be useful for testing other potential macrophage modulators in vivo.

Thermoresponsive nanoparticle agglomeration/aggregation in salt solutions: Dependence on graft density.

Gold nanoparticles with a graft density of 0.09, 0.30 and 0.40chains/nm2 of poly(N-isopropylacrylamide) were reproducibly synthesized by varying the ratio of disulfide terminated poly(N-isopropylacrylamide) to gold nanoparticle. The polymer coated nanoparticles were stable at room temperature in 50mM NaCl, yet agglomerated at 37°C. Previous studies have observed conflicting results as to the reversibility of this agglomeration. Particle agglomeration with three different graft densities was studied in 50mM NaCl by measurements of their localized surface plasmon resonance and hydrodynamic diameter, and imaging with electron microscopy. Agglomerates with a polymer graft density of 0.30 and 0.40chains/nm2 could be dispersed with sonication, while particles with a graft density of 0.09chains/nm2 irreversibly aggregated. The graft density dependence on whether agglomeration or aggregation occurred is due to changes in collapsed polymer steric effects. Localized surface plasmon resonance measurements of agglomerates were discordant with hydrodynamic diameter measurements in determining agglomeration reversibility, which shed light on reasons previous reports yielded different interpretations on the reversibility of this agglomeration. This work demonstrates how polymer graft density affects thermoresponsive nanoparticle stability in salt solutions and the need for use of complementary techniques when determining agglomeration.

Effects of delayed delivery of dexamethasone-21-phosphate via subcutaneous microdialysis implants on macrophage activation in rats.

Macrophage activation is of interest in the biomaterials field since macrophages with an M(Dex) characteristic phenotype, i.e., CD68(+)CD163(+), are believed to result in improved integration of the biomaterial as well as improved tissue remodeling and increased biomaterial longevity. To facilitate delivery of a macrophage modulator, dexamethasone-21-phosphate (Dex), microdialysis probes were subcutaneously implanted in male Sprague-Dawley rats. Dex localized delivery was delayed to the third day post implantation as a means to alter macrophage activation state at an implant site. To better elucidate the molecular mechanisms associated with M(Dex) macrophage activation, CCL2 was quantified in dialysates, gene expression ratios were determined from excised tissue surrounding the implant, histological analyses, and immunohistochemical analyses (CD68, CD163) were performed. Delayed Dex infusion resulted in the up-regulation of IL-6 at the transcript level in the tissue in contact with the microdialysis probe and decreased CCL2 concentrations collected in dialysates. Histological analyses showed increased cellular density as compared to controls in response to delayed Dex infusion. Dex delayed infusion resulted in an increased percentage of CD68(+)CD163(+), M(Dex), macrophages in the tissue surrounding the microdialysis probe as compared to probes that served as controls.

A review of flux considerations for in vivo neurochemical measurements.

The mass transport or flux of neurochemicals in the brain and how this flux affects chemical measurements and their interpretation is reviewed. For all endogenous neurochemicals found in the brain, the flux of each of these neurochemicals exists between sources that produce them and the sites that consume them all within µm distances. Principles of convective-diffusion are reviewed with a significant emphasis on the tortuous paths and discrete point sources and sinks. The fundamentals of the primary methods of detection, microelectrodes and microdialysis sampling of brain neurochemicals are included in the review. Special attention is paid to the change in the natural flux of the neurochemicals caused by implantation and consumption at microelectrodes and uptake by microdialysis. The detection of oxygen, nitric oxide, glucose, lactate, and glutamate, and catecholamines by both methods are examined and where possible the two techniques (electrochemical vs. microdialysis) are compared. Non-invasive imaging methods: magnetic resonance, isotopic fluorine MRI, electron paramagnetic resonance, and positron emission tomography are also used for different measurements of the above-mentioned solutes and these are briefly reviewed. Although more sophisticated, the imaging techniques are unable to track neurochemical flux on short time scales, and lack spatial resolution. Where possible, determinations of flux using imaging are compared to the more classical techniques of microdialysis and microelectrodes.

Bioanalytical chemistry of cytokines--a review.

Cytokines are bioactive proteins produced by many different cells of the immune system. Due to their role in different inflammatory disease states and maintaining homeostasis, there is enormous clinical interest in the quantitation of cytokines. The typical standard methods for quantitation of cytokines are immunoassay-based techniques including enzyme-linked immusorbent assays (ELISA) and bead-based immunoassays read by either standard or modified flow cytometers. A review of recent developments in analytical methods for measurements of cytokine proteins is provided. This review briefly covers cytokine biology and the analysis challenges associated with measurement of these biomarker proteins for understanding both health and disease. New techniques applied to immunoassay-based assays are presented along with the uses of aptamers, electrochemistry, mass spectrometry, optical resonator-based methods. Methods used for elucidating the release of cytokines from single cells as well as in vivo collection methods are described.

Localized delivery of dexamethasone-21-phosphate via microdialysis implants in rat induces M(GC) macrophage polarization and alters CCL2 concentrations.

Microdialysis sampling probes were implanted into the subcutaneous space on the dorsal side of male Sprague Dawley rats to locally deliver dexamethasone-21-phosphate (Dex) with the aim of altering in vivo macrophage polarization. Macrophage polarization is of significant interest in the field of biomaterials since wound-healing macrophages are a possible means to extend implant life as well as improve tissue remodeling to an implant. Quantitative analysis of CCL2 in collected dialysates, gene expression and immunohistochemistry performed on the tissue surrounding the microdialysis implant were used to evaluate if Dex polarized macrophages. Dex infusion down-regulated IL-6 and CCL2 gene expression and decreased CCL2 concentrations in dialysates collected at the implant site. Dex appeared to have no significant effect on the gene regulation of CD163, a commonly used M2c macrophage surface marker; Arg2; and iNOS2. However, Dex infusion was effective at increasing the number of CD163(+) cells surrounding the implanted microdialysis probe. This work demonstrates the use of microdialysis sampling to deliver agents such as Dex to alter macrophage polarization in vivo while allowing the ability to collect cytokines in the surrounding microenvironment.

In vivo microdialysis sampling of adipokines CCL2, IL-6, and leptin in the mammary fat pad of adult female rats.

Adipocytes from white adipose tissue secrete cytokines and other bioactive proteins which are collectively termed adipokines. Adiposity has been linked with increased breast cancer risk as adipokines secreted by adipocytes significantly affect epithelial cells from which breast cancer arises. Measurement of extracellular adipokine concentrations that would be involved in signaling through mammary tissue is therefore of importance. In this work, microdialysis sampling was used to collect adipokines from the interstitial space of the mammary fat pad of female rats under isoflurane anesthesia. The adipokines CCL2 (MCP-1), leptin and IL-6 were quantified from dialysate samples and compared to total tissue concentrations surrounding the implanted probes. After three hours of microdialysis sampling at 1 µL min(-1), the respective median values for these adipokines in dialysate samples were approximately 175 pg mL(-1) (CCL2), 150 pg mL(-1) (IL-6) and 750 pg mL(-1) (leptin). Adipokine protein levels from dialysates were an order of magnitude lower than levels obtained directly from mammary tissue. However, the adipokine concentrations between excised tissue surrounding the microdialysis sampling probes and control tissue without implants did not differ. This work demonstrates the utility of microdialysis sampling to quantify mammary gland adipokine levels, with relevance to understanding mammary physiology.

Microdialysis sampling techniques applied to studies of the foreign body reaction.

Implanted materials including drug delivery devices and chemical sensors undergo what is termed the foreign body reaction (FBR). Depending on the device and its intended application, the FBR can have differing consequences. An extensive scientific research effort has been devoted to elucidating the cellular and molecular mechanisms that drive the FBR. Important, yet relatively unexplored, research includes the localized tissue biochemistry and the chemical signaling events that occur throughout the FBR. This review provides an overview of the mechanisms of the FBR, describes how the FBR affects different implanted devices, and illustrates the role that microdialysis sampling can play in further elucidating the chemical communication processes that drive FBR outcomes.

Comparison of microdialysis sampling perfusion fluid components on the foreign body reaction in rat subcutaneous tissue.

Microdialysis sampling is a commonly used technique for collecting solutes from the extracellular space of tissues in laboratory animals and humans. Large molecular weight solutes can be collected using high molecular weight cutoff (MWCO) membranes (100kDa or greater). High MWCO membranes require addition of high molecular weight dextrans or albumin to the perfusion fluid to prevent fluid loss via ultrafiltration. While these perfusion fluid additives are commonly used during microdialysis sampling, the tissue response to the loss of these compounds across the membrane is poorly understood. Tissue reactions to implanted microdialysis sampling probes containing different microdialysis perfusion fluids were compared over a 7-day time period in rats. The base perfusion fluid was Ringer's solution supplemented with either bovine serum albumin (BSA), rat serum albumin (RSA), Dextran-70, or Dextran-500. A significant inflammatory response to Dextran-70 was observed. No differences in the tissue response between BSA and RSA were observed. Among these agents, the BSA, RSA, and Dextran-500 produced a significantly reduced inflammatory response compared to the Dextran-70. This work demonstrates that use of Dextran-70 in microdialysis sampling perfusion fluids should be eliminated and replaced with Dextran-500 or other alternatives.

In vivo microdialysis sampling of cytokines from rat hippocampus: comparison of cannula implantation procedures.

Cytokines are signaling proteins that have been of significant importance in the field of immunology, since these proteins affect different cells in the immune system. In addition to their immune system significance, these proteins have recently been referred to as a third chemical communication network within the CNS. The role that cytokines play in orchestrating the immune response within tissues after a mechanical injury leads to potential complications if the source of cytokines (i.e., trauma vs disease) is of interest. Microdialysis sampling has seen wide use in collection of many different solutes within the CNS. Yet, implantation of microdialysis guide cannulas and the probes creates tissue injury. In this study, we compared the differences in cytokine levels in dialysates from 4 mm, 100 kDa molecular weight cutoff (MWCO) polyethersulfone membrane microdialysis probes implanted in the hippocampus of male Sprague-Dawley rats. Comparisons were made between animals that were dialyzed immediately after cannula implantation (day 0), 7 days post cannula implantation (day 7), and repeatedly sampled on day 0 and day 7. Multiplexed bead-based immunoassays were used to quantify CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CXCL1 (KC/GRO), CXCL2 (MIP-2), IL-1ß, IL-6, and IL-10 in dialysates. Differences in cytokine concentrations between the different treatment groups were observed with higher levels of inflammatory cytokines measured in day 7 cannulated animals. Only CCL3 (MIP-1α), CXCL1 (KC/GRO), CXCL2 (MIP-2), and IL-10 were measured above the assay limits of detection for a majority of the dialysates, and their concentrations were typically in the low to high (10-1000) picogram per milliliter range. The work described here lays the groundwork for additional basic research studies with microdialysis sampling of cytokines in rodent CNS.

Modulation of the foreign body reaction for implants in the subcutaneous space: microdialysis probes as localized drug delivery/sampling devices.

Modulation of the foreign body reaction is considered to be an important step toward creation of implanted sensors with reliable long-term performance. In this work, microdialysis probes were implanted into the subcutaneous space of Sprague-Dawley rats. The probe performance was evaluated by comparing collected endogenous glucose concentrations with internal standard calibration (2-deoxyglucose, antipyrine, and vitamin B12). Probes were tested until failure, which for this work was defined as loss of fluid flow. In order to determine the effect of fibrous capsule formation on probe function, monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) was delivered locally via the probe to increase capsule thickness and dexamethasone 21-phosphate was delivered to reduce capsule thickness. Probes delivering MCP-1 had a capsule that was twice the thickness (500-600 µm) of control probes (200-225 µm) and typically failed 2 days earlier than control probes. Probes delivering dexamethasone 21-phosphate had more fragile capsules and the probes typically failed 2 days later than controls. Unexpectedly, extraction efficiency and collected glucose concentrations exhibited minor differences between groups. This is an interesting result in that the foreign body capsule formation was related to the duration of probe function but did not consistently relate to probe calibration.

Antibody-enhanced microdialysis collection of CCL2 from rat brain.

Chemokine(C-C motif) Ligand 2 (CCL2 or MCP-1) is a signaling protein that is released under various conditions. In this study we demonstrate the first microdialysis collection of CCL2 from rat brain tissue using antibody-enhanced microdialysis. A monoclonal antibody to CCL2 was included in the dialysis perfusion fluid as an affinity agent to enhance the recovery of CCL2 both in vitro and in vivo. In vitro it was found that the use of antibody affinity agent increases the relative recovery of CCL2 from 9.6±3.4% to 37.5±10.2% and 64.8±11.7% (n=10) at flow rates of 2µL/min and 1µL/min, respectively. Following the in vitro observation, CCL2 was collected from rat brain with microdialysis sampling using both control and antibody-included perfusion fluids. The in vivo data showed that relative recovery was increased at all but the first time point. This shows that the use of free antibody in the perfusion fluid increases the relative recovery of CCL2 and this enhanced microdialysis method may be applicable to other cytokines.

Heparin-immobilized microspheres for the capture of cytokines.

The preparation and characterization of heparin-immobilized microspheres which were used to bind acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1/CCL2), and regulation upon activation normal T cell express sequence (RANTES/CCL5) is described. These beads were used as trapping agents in microdialysis sampling experiments in a separate study. Both free heparin and a synthesized heparin-albumin conjugate were immobilized onto microspheres and compared for their effectiveness. The heparin-albumin conjugate microspheres exhibited significant nonspecific adsorption which appeared to be due to the albumin content. The prepared heparin-immobilized microspheres were stable for 3 months at 4 °C. A bead-based flow cytometric assay was developed to study the binding capacity and specificity of the heparin-immobilized microspheres to cytokines. These heparin-immobilized microspheres exhibited broad dynamic ranges for binding to the four cytokines (aFGF, 1.0-1,000 ng/mL; VEGF, 0.5-1,000 ng/mL; CCL2, 1.95-1,000 ng/mL; CCL5, 1.95-500 ng/mL). Fast binding kinetics of the cytokines to the heparin-immobilized beads suggests that these beads may be useful as affinity agents in microfluidic flow systems.

Long-term subcutaneous microdialysis sampling and qRT-PCR of MCP-1, IL-6 and IL-10 in freely-moving rats.

Cytokines are important mediators of the wound healing response. However, sampling of cytokines from the interstitial fluid at a healing wound site in experimental animals is a challenge. Microdialysis sampling is an in vivo collection option for this purpose as it permits continuous sampling, while remaining contiguous with the wound microenvironment. The polymeric membrane of the microdialysis probe is a foreign material thus allowing a unique approach to sample cytokines generated during a foreign body response (FBR). The focus of these studies was to use microdialysis sampling to collect cytokines from a microdialysis probe implant site in a rat model of a FBR up to 6 days post implantation. Fluorescent bead-based immunoassays (Luminex™) were used to quantify monocyte chemoattractant protein-1 (MCP-1/CCL2), interleukin-6 (IL-6) and interleukin-10 (IL-10) in the dialysates. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to cross validate the protein measurements obtained via micorodialysis sampling. A histological examination of tissue was also performed to assess the progression in leukocyte extravasation and collagen deposition surrounding implanted probes. Our findings demonstrate that in vivo microdialysis sampling can be used to collect temporal concentrations of cytokines which are consistent with wound healing and the development of a FBR.

In vitro and in vivo affinity microdialysis sampling of cytokines using heparin-immobilized microspheres.

Heparin-immobilized microspheres were included in microdialysis sampling perfusion fluids under both in vitro and in vivo conditions to improve the recovery of different cytokines, acidic fibroblast growth factor, vascular endothelial growth factor, monocyte chemoattractant protein-1 (or CCL2), and regulation upon activation normal T cell express sequence (or CCL5). Different strategies to dissociate captured CCL2 and CCL5 from the immobilized heparin were attempted, and both cytokines could be quantitatively eluted from the beads using a phosphate buffer (pH 7.4) containing 25% (v/v) acetonitrile which did not interfere with the subsequent detection of cytokine using an ELISA assay. Using these heparin-immobilized microspheres, a two to fivefold increase of microdialysis relative recovery (RR) was achieved for the four cytokines from a quiescent solution. Enhanced microdialysis RR of CCL2 using the heparin-immobilized microspheres from microdialysis probes implanted into the peritoneal cavity of a rat was performed to test the in vivo application. This work suggests that the heparin-immobilized microspheres provide an alternative affinity agent to the previously used antibody-immobilized microspheres for enhanced microdialysis sampling of cytokines.