Feasibility of the Ussing chamber technique for the determination of in vitro jejunal permeability of passively absorbed compounds in different animal species.

The aim of this study was to assess the feasibility of the Ussing chamber technique for the determination of the jejunal permeability of passively absorbed, high permeability model compounds (acetaminophen and ketoprofen) in different animal species. Additionally, electrophysiological measurements and histological examination of pre- and post-incubation tissue specimens were performed. Apparent permeability coefficients of turkey and dog jejunum were low and highly variable due to tissue fragility caused by differences in thickness of the remaining intestinal layers after stripping and resulting in severe damage. Pig and horse jejunum were markedly more suitable for permeability determinations and mild signs of deterioration were noticed after 120 min of incubation. Transepithelial electrical resistance and potential difference did not correlate well with the observed tissue damage. From these data, the Ussing chamber technique appears to allow for permeability measurements within a species, but seems unsuitable for interspecies permeability comparison. However, further validation of the method with low permeability compounds and actively transported compounds is needed.

Microenvironment array chip for cell culture environment screening.

We have developed a microarray of cell culture environments composed of a combination of soluble factors and extracellular matrices for screening of cell culture environment.

The membrane chamber: a new type of in vitro recording chamber.

In vitro brain slice electrophysiology is a powerful and highly successful technique where a thin slice is cut from the brain and kept alive artificially in a recording chamber. The design of this recording chamber is pivotal to the success and the quality of such experiments. Most often one of two types of chambers is used today, the interface chamber or the submerged chamber. These chambers, however, have the disadvantage that they are limited in either their experimental or their physiological properties respectively. Here we present a new working principle for an in vitro chamber design which aims at combining the advantages of the classical designs whilst overcoming their disadvantages. This is achieved by using a semipermeable membrane on which the slice is placed. The membrane allows for a fast flow of artificial cerebrospinal fluid of up to at least 17 ml/min. Due to a Bernoulli effect, this high speed flow also causes a 64% increase in flow of solution across the membrane on which the slice rests. The fact that the membrane is transparent introduces the possibility of wide field inverted optical imaging to brain slice electrophysiology. The utility of this setup was demonstrated in the recording of local field potential, single cell and voltage sensitive dye imaging data simultaneously from an area smaller then 1/8mm(2). The combination of all these features in the membrane chamber make it a versatile and promising device for many current and future in vitro applications, especially in the regard to optical imaging.

Autologous growth factors versus autogenous graft for anterior cervical interbody fusion: an in vivo caprine model.

OBJECT: Using an in vivo caprine model, authors in this study compared the efficacy of autologous growth factors (AGFs) with autogenous graft for anterior cervical interbody arthrodesis. METHODS: Fourteen skeletally mature Nubian goats were used in this study and followed up for a period of 16 weeks postoperatively. Anterior cervical interbody arthrodesis was performed at the C3-4 and C5-6 vertebral levels. Four interbody treatment groups (7 animals in each group) were equally randomized among the 28 arthrodesis sites: Group 1, autograft alone; Group 2, autograft + cervical cage; Group 3, AGFs + cervical cage; and Group 4, autograft + anterior cervical plate. Groups 1 and 4 served as operative controls. Autologous growth factors were obtained preoperatively from venous blood and were ultra-concentrated. Following the 16-week survival period, interbody fusion success was evaluated based on radiographic, biomechanical, and histological analyses. RESULTS: All goats survived surgery without incidence of vascular or infectious complications. Radiographic analysis by 3 independent observers indicated fusion rates ranging from 9 (43%) of 21 in the autograft-alone and autograft + cage groups to 12 (57%) of 21 in the autograft + anterior plate group. The sample size was not large enough to detect any statistical significance in these observed differences. Biomechanical testing revealed statistical differences (p < 0.05) between all treatments and the nonoperative controls under axial rotation and flexion and extension loading. Although the AGF + cage and autograft-alone treatments appeared to be statistically different from the intact spine during lateral bending, larger variances and smaller relative differences precluded a determination of statistical significance. Histomorphometric analysis of bone formation within the predefined fusion zone indicated quantities of bone within the interbody cage ranging from 21.3 +/- 14.7% for the AGF + cage group to 34.5 +/- 9.9% for the autograft-alone group. CONCLUSIONS: The results indicated no differences in biomechanical findings among the treatment groups and comparable levels of trabecular bone formation within the fusion site between specimens treated with autogenous bone and those filled with the ultra-concentrated AGF extract. In addition, interbody cage treatments appeared to maintain disc space height better than autograft-alone treatments.

Remote switching of temperature, gaseous, and aqueous phase in a low-volume interface chamber for brain slices.

A new remote-controlled interface-type chamber was designed in order to conduct experiments in brain slices involving gas, fluid, and temperature changes with as little tissue manipulation as possible. The chamber allows for extremely quick changes between different fluid and/or gaseous phases and for active cooling as well as heating by using a set of electromechanical valves and Peltier elements. The design drawings are complemented by exemplary tests of temperature and gas changes, and electrophysiological recordings of slices manipulated with gas and fluid alterations were used to test the efficacy and accuracy of the design. Changing between normoxia and anoxia needs less than 30 s, while the readjustment of the chamber to a new, preset temperature is accomplished in about 1 min. Supplementary data provide a proposal for the electronic circuit diagram. This chamber design should simplify data acquisition in interface environments.

A microfluidic brain slice perfusion chamber for multisite recording using penetrating electrodes.

To analyze the spatiotemporal dynamics of network activity in a brain tissue slice, it is useful to record simultaneously from multiple locations. When obtained from laminar structures such as the hippocampus or neocortex, multisite recordings also yield information about subcellular current distributions via current source density analysis. Multisite probes developed for in vivo recordings could serve these purposes in vitro, allowing recordings to be obtained from brain slices at sites deeper within the tissue than currently available surface recording methods permit. However, existing recording chambers do not allow for the insertion of lamina-spanning probes that enter through the edges of brain slices. Here, we present a novel brain slice recording chamber design that accomplishes this goal. The device provides a stable microfluidic perfusion environment in which tissue health is optimized by superfusing both surfaces of the slice. Multichannel electrodes can be inserted parallel to the surface of the slice, at any depth relative to the surface. Access is also provided from above for the insertion of additional recording or stimulating electrodes. We illustrate the utility of this recording configuration by measuring current sources and sinks during theta burst stimuli that lead to the induction of long-term potentiation in hippocampal slices.

Usefulness of polyetheretherketone (PEEK) cage with plate augmentation for anterior arthrodesis in traumatic cervical spine injury.

BACKGROUND CONTEXT: Even though many clinical reports about cages have been documented in patients with degenerative disorders, reports were scarce for traumatic injury cases, and those cases using metal cages were restricted to only one-level injury. PURPOSE: To evaluate the usefulness of polyetheretherketone (PEEK) cage and plate construction in anterior interbody fusions (AIF) for traumatic cervical spine injuries by analyzing radiographic changes and clinical outcomes. STUDY DESIGN/SETTING: Retrospective study. PATIENT SAMPLE: Fifty-eight patients (91 levels) underwent cage and plate construction for treatment of traumatic cervical spine injury. OUTCOME MEASURES: The fusion rate, fusion time, changes of Cobb angle, subsidence rate, and adjacent level changes were assessed as a radiographic outcome. Clinical analysis includes the recovery rate on the American Spinal Injury Association (ASIA) impairment scale and the presence of the complications. METHODS: We evaluated 58 patients (91 levels) who underwent surgery and had at least 24 months in follow-up study. Radiographic evaluation included the assessment of interbody fusion rate, fusion time, changes of Cobb angle, subsidence rate, and adjacent level changes. Clinical assessment was done by analyzing recovery state of ASIA impairment scale from preoperative period to the last follow-up and by evaluating complications. RESULTS: Fifty-four cases showed bony fusion within 3 months after the surgery. The mean Cobb angle between the vertebral bodies was 2.54 degrees before operation, 9.13 degrees after operation, and 8.39 degrees at the latest follow-up. The mean intervertebral disc height was increased by 3.01 mm after the operation, but the mean height was 2.17 mm shorter at the last follow-up than after postoperation. In terms of clinical results, five Grade A cases and one Grade B case as assessed by the ASIA impairment scale were unchanged until the last follow-up. Twenty-three cases of Grade C, 16 cases of Grade D, and 13 cases of Grade E improved to seven cases, 26 cases, and 19 cases, respectively. Three cases went through additional surgery, two posterior fusions for delayed union and posterior instability and one AIF for adjacent level disease. CONCLUSION: The PEEK cage and additional plate fixation is a surgical procedure that decreases donor site morbidity, obtains high fusion rate with rigid fixation, and provides satisfactory clinical outcome for traumatic cervical spine injuries, regardless of the numbers of the involved levels.

A device to facilitate preparation of high-density neural cell cultures in MEAs.

A device to facilitate high-density seeding of dissociated neural cells on planar multi-electrode arrays (MEAs) is presented in this paper. The device comprises a metal cover with two concentric cylinders-the outer cylinder fits tightly on to the external diameter of a MEA to hold it in place and an inner cylinder holds a central glass tube for introducing a cell suspension over the electrode area of the MEA. An O-ring is placed at the bottom of the inner cylinder and the glass tube to provide a fluid-tight seal between the glass tube and the MEA electrode surface. The volume of cell suspension in the glass tube is varied according to the desired plating density. After plating, the device can be lifted from the MEA without leaving any residue on the contact surface. The device has enabled us to increase and control the plating density of neural cell suspension with low viability, and to prepare successful primary cultures from cryopreserved neurons and glia. The cultures of cryopreserved dissociated cortical neurons that we have grown in this manner remained spontaneously active over months, exhibited stable development and similar network characteristics as reported by other researchers.

Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability.

Brain slice preparations are well-established models for a wide spectrum of in vitro investigations in the neuroscience discipline. However, these investigations are limited to acute preparations or thin organotypic culture preparations due to the lack of a successful method that allows culturing of thick organotypic brain slices. Thick brain slice cultures suffer necrosis due to ischemia deep in the tissue resulting from a destroyed circulatory system and subsequent diffusion-limited supply of nutrients and oxygen. Although thin organotypic brain slice cultures can be successfully cultured using a well-established roller-tube method (a monolayer organotypic culture) (Gahwiler B H. Organotypic monolayer cultures of nervous tissue. J Neurosci Methods. 1981; 4: 329-342) or a membrane-insert method (up to 1-4 cell layers, <150 microm) (Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of neural tissue. J Neurosci Methods 1991; 37: 173-182), these methods fail to support thick tissue preparations. A few perfusion methods (using submerged or interface/microfluidic chambers) have been reported to enhance the longevity (up to few hours) of acute slice preparations (up to 600 microm thick) (Hass HL, Schaerer B, Vosmansky M. A simple perfusion chamber for study of nervous tissue slices in vitro. J Neurosci Methods 1979; 1: 323-325; Nicoll RA, Alger BE. A simple chamber for recording from submerged brain slices. J Neurosci Methods 1981; 4: 153-156; Passeraub PA, Almeida AC, Thakor NV. Design, microfabrication and characterization of a microfluidic chamber for the perfusion of brain tissue slices. J Biomed Dev 2003; 5: 147-155). Here, we report a unique interstitial microfluidic perfusion technique to culture thick (700 microm) organotypic brain slices. The design of the custom-made microperfusion chamber facilitates laminar, interstitial perfusion of oxygenated nutrient medium throughout the tissue thickness with concomitant removal of depleted medium and catabolites. We examined the utility of this perfusion method to enhance the viability of the thick organotypic brain slice cultures after 2 days and 5 days in vitro (DIV). We investigated the range of amenable flow rates that enhance the viability of 700 microm thick organotypic brain slices compared to the unperfused control cultures. Our perfusion method allows up to 84.6% viability (p<0.01) and up to 700 microm thickness, even after 5 DIV. Our results also confirm that these cultures are functionally active and have their in vivo cyto-architecture preserved. Prolonged viability of thick organotypic brain slice cultures will benefit scientists investigating network properties of intact organotypic neuronal networks in a reliable and repeatable manner.

Biomechanical rigidity of an all-polyetheretherketone anterior thoracolumbar spinal reconstruction construct: an in vitro corpectomy model.

BACKGROUND CONTEXT: Polyetheretherketone (PEEK) is gaining favor as a spinal implant material for interbody and corpectomy cages as well as stabilizing rods. However, there has been little correlation to a relevant and reproducible clinical model. Biomechanical data on PEEK rod constructs have not been reported. PURPOSE: To quantify the stabilizing effects of PEEK versus titanium (Ti) instrumentation in a thoracolumbar corpectomy model. STUDY DESIGN: Corpectomy and randomized instrumentation with an all-Ti, all-PEEK, and hybrid cage/rod construct were performed on cadaveric spines to assess biomechanical differences. METHODS: Pure unconstrained bending moments were applied to the intact spine and subsequent test constructs in the three physiologic planes using a load control protocol. Motion tracking and analysis were carried out to quantify and compare the range of motion (ROM) between different test constructs in each plane. RESULTS: Flexion ROM did not show significant changes compared with intact, whereas the all-Ti and hybrid construct reduced ROM significantly in extension. Lateral bending was significantly reduced in all the treatment groups. Rotational stability of the construct was significantly compromised by an all-PEEK spinal construct. CONCLUSION: The rigidity of the corpectomy construct increased as the amount of Ti in the construct increased. A hybrid construct incorporating a PEEK corpectomy cage and Ti rods may provide adequate stability for an anterior thoracolumbar reconstruction in the sagittal and coronal planes. An all-PEEK construct may provide adequate stability in the coronal and sagittal planes but may compromise the stability significantly in axial rotation. Consideration should be given for supplemental posterior instrumentation if an all-PEEK construct is used in an anterior thoracolumbar spinal reconstruction procedure.

A new model for chronic in vivo analysis of the periosteal microcirculation.

OBJECTIVE: Microvascular perfusion is indispensable for the growth and remodulation of membrane bone. Trauma, inflammation and surgical interventions may alter periosteal perfusion. However, there is not much known about periosteal perfusion in membrane bones. Therefore, the aim of this study was to establish a new chronic model that permits the repeated in vivo analysis of the microcirculation of periosteum. METHODS: A circular skin island with a diameter of 6 mm was excised at the forehead of six Lewis rats. Then a newly developed chamber was implanted, containing a coverglass to protect the periosteum. Intravital microscopy (IVM) enables a quantitative analysis of periosteal microcirculation immediately as well as 3, 5 and 10 days after chamber implantation. At the end of the experiment the calvaria and periosteum were removed for histological examination. Six unoperated Lewis rats served as histological controls. RESULTS: The periosteal microcirculation remained stable over 10 days. The implantation of the chamber did not result in any substantial inflammatory response. The functional microvascular density was 131.2+/-19.3 cm/cm(2). The histological examinations revealed a regular anatomical structure of periosteum and bone including an intact interface. CONCLUSION: The presented model allows for the first time to conduct a repetitive, quantitative in vivo analysis of the periosteal microcirculation in membrane bone. Future studies may thus evaluate novel strategies to influence the periosteal perfusion.

An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.

The accessory olfactory system (AOS) in mammals detects and processes information from liquid-phase environmental odorants, including pheromones. The AOS carries out tasks such as individual recognition, learning, and decision-making with relatively few stages of neural processing; it thus represents an attractive system for investigating the neural circuits that carry out these functions. Progress in understanding the AOS has long been impeded by its relative inaccessibility to standard physiological approaches. In this report, we detail a novel dissection and tissue perfusion strategy that improves access to the accessory olfactory bulb (AOB) while maintaining afferent connections from sensory neurons in the vomeronasal organ (VNO). Mitral cells demonstrated spontaneous and evoked firing patterns consistent with recent in vivo reports. We assayed cell degradation in the AOB tissue using Fluoro-Jade C and found that the VNO and AOB glomerular, external plexiform, and mitral cell layers showed minimal signs of degeneration for up to 6h. Whereas histology indicated some degeneration in the deep inhibitory granule cell layer over time, electrophysiological assays demonstrated intact inhibitory function on mitral cells. Pharmacological blockade of GABA(A) receptors with 3microM SR95531 (gabazine) resulted in increased evoked mitral cell activity. Furthermore, mitral cells displayed suppression of responses to preferred urine stimuli when preferred and non-preferred stimuli were mixed, an effect thought to involve functional laterally connected inhibition. These results demonstrate the utility of whole mount ex vivo preparations for studying sensory processing in the AOS, and suggest that similar strategies may improve experimental access to other difficult-to-study neural circuits.

Pressure-driven perfusion culture microchamber array for a parallel drug cytotoxicity assay.

This article reports a pressure-driven perfusion culture chip developed for parallel drug cytotoxicity assay. The device is composed of an 8 x 5 array of cell culture microchambers with independent perfusion microchannels. It is equipped with a simple interface for convenient access by a micropipette and connection to an external pressure source, which enables easy operation without special training. The unique microchamber structure was carefully designed with consideration of hydrodynamic parameters and was fabricated out of a polydimethylsiloxane by using multilayer photolithography and replica molding. The microchamber structure enables uniform cell loading and perfusion culture without cross-contamination between neighboring microchambers. A parallel cytotoxicity assay was successfully carried out in the 8 x 5 microchamber array to analyze the cytotoxic effects of seven anticancer drugs. The pressure-driven perfusion culture chip, with its simple interface and well-designed microfluidic network, will likely become an advantageous platform for future high-throughput drug screening by microchip.

Rapid prototyping for neuroscience and neural engineering.

Rapid prototyping (RP) is a useful method for designing and fabricating a wide variety of devices used for neuroscience research. The present study confirms the utility of using fused deposition modeling, a specific form of RP, to produce three devices commonly used for basic science experimentation. The accuracy and precision of the RP method varies according to the type and quality of the printer as well as the thermoplastic substrate. The printer was capable of creating device channels with a minimum diameter of 0.4 or 0.6mm depending on the orientation of fabrication. RP enabled the computer-aided design and fabrication of three custom devices including a cortical recording/stroke induction platform capable of monitoring electrophysiological function during ischemic challenge. In addition to the recording platform, two perfusion chambers and a cranial window device were replicated with sub-millimeter precision. The ability to repeatedly modify the design of each device with minimal effort and low turn-around time is helpful for oft-unpredictable experimental conditions. Results obtained from validation studies using both the cortical recording platform and perfusion chamber did not vary from previous results using traditional hand-fabricated or commercially available devices. Combined with computer-aided design, rapid prototyping is an excellent alternative for developing and fabricating custom devices for neuroscience research.

Conical electron tomography of a chemical synapse: polyhedral cages dock vesicles to the active zone.

In this study, we tested the hypothesis that the structure of the active zone of chemical synapses has remained uncertain because of limitations of conventional electron microscopy. To resolve these limitations, we reconstructed chemical synapses of rat neocortex, the archetypical "average" synapse, by conical electron tomography, a method that exhibits an isotropic in plane resolution of approximately 3 nm and eliminates the need to impose symmetry or use averaging methods to increase signal-to-noise ratios. Analysis of 17 reconstructions by semiautomatic density segmentation indicated that the active zone was constructed of a variable number of distinct "synaptic units" comprising a polyhedral cage and a corona of approximately seven vesicles. The polyhedral cages measured approximately 60 nm in diameter, with a density of approximately 44/microm2 and were associated with vesicles at the active zone ("first tier"). Vesicles in this first-tier position represented approximately 7.5% of the total number of vesicles in the terminal and were contiguous, hemifused (approximately 4% of total), or fully fused (approximately 0.5% of total) to the plasma membrane. Our study supports the hypothesis that rat neocortical synapses are constructed of variable numbers of distinct synaptic units that facilitate the docking of vesicles to the active zone and determine the number of vesicles available for immediate release.

A reversed-phase ion-interaction chromatographic method for in-vitro estimation of the percutaneous absorption of water-soluble UV filters.

An analytical method based on ion-interaction chromatography with UV detection for simultaneous in-vitro estimation of the percutaneous absorption of the most used water-soluble UV filters in sunscreen cosmetics is proposed. These UV filters were phenylbenzimidazole sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, benzophenone-4, and terephthalylidene dicamphor sulfonic acid. The methodology is based on applying the sunscreen containing the target UV filters to human epidermis in a diffusion cell. Analytes are determined in the receptor solution. To ensure skin integrity, screening of the cells was carried out by analytical determination of a marker. Analytical variables such as percentage ethanol, concentration of ion-pairing agent, pH of the mobile phase, and temperature were studied in order to achieve high resolution of the chromatographic peaks in the lowest possible time of analysis. The conditions selected consisted of a mobile phase composed of 35:65 (v/v) ethanol-ammonium acetate buffer solution (pH 4, containing 50 mmol L(-1) tetra-n-butylammonium bromide). The chromatographic determination was carried out with the analytical column at 50 degrees C. UV detection was carried out at the maximum absorption wavelength for each analyte. The limit of detection (3s(y/x)/b) ranged from 16 to 65 ng mL(-1), depending on the analyte.

A removable silicone elastomer seal reduces granulation tissue growth and maintains the sterility of recording chambers for primate neurophysiology.

The maintenance of the sterility of craniotomies for serial acute neurophysiological recordings is exacting and time consuming yet is vital to the health of valuable experimental animals. We have developed a method to seal the craniotomy with surgical grade silicone elastomer (Silastic) in a hermetically sealed chamber. Under these conditions the tissues in the craniotomy and the inside surface of the chamber remain unpopulated by bacteria. The silicone elastomer sealant retarded the growth of granulation tissue on the dura and reduced the procedures required to maintain ideal conditions for neurophysiological recordings.

Transdermal penetration of UV filters.

A penetration study of 2-ethylhexyl-4-methoxycinnamate (EHMC), 4-methyl benzylidenecamphor (MBC), butyl methoxydibenzoylmethane (BMBM), 2-ethylhexyl-2,4,5-trimethoxycinnamate (EHTMC) and di(2-ethylhexyl)-2,4,5-trimethoxybenzalmalonate (TMB) through baby mouse skin (Mus musculus Linn.) was carried out using a vertical Franz diffusion cell. At 4.4 mg/cm(2) coverage of UV filter on the skin, 2.98 +/- 0.38, 1.15 +/- 0.14 and 0.80 +/- 0.28% of the applied EHMC, MBC and BMBM were detected in the receptor fluid at 24 h after application. Penetrations of UV filter in an ethanolic solution and lotion forms were comparable. EHTMC and TMB showed insignificant penetration across the baby mouse skins. Baby mouse skins kept at 4, -20 and -80 degrees C gave similar EHMC penetration results. Penetrations of EHMC, BMBM, EHTMC and TMB across human epidermis were carried out upon 5 volunteers using the suction blister technique. The results also confirmed the significant penetrations of EHMC and BMBM and the insignificant penetrations of EHTMC and TMB.

Incubation of environmental samples in a diffusion chamber increases the diversity of recovered isolates.

The majority of microorganisms from natural environments cannot be grown in the laboratory. The diffusion-chamber-based approach is an alternative method that allows microorganisms to grow in their natural environment. An inoculum is sandwiched between semipermeable (0.03-mum-pore-size) membranes of the chamber, which is then returned to the source environment. The chamber allows for a free exchange of chemicals with the external milieu by diffusion while restricting the movement of cells. We used freshwater pond sediment to inoculate diffusion chambers and petri dishes. The diffusion chambers were incubated on top of the sediment for 4 weeks. Both chamber and petri dish cultivation resulted in the isolation of numerous representatives of Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. However, the diffusion-chamber-based approach also led to the isolation of species from rarely cultivated groups, such as Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and Acidobacteria. Material from the chambers was also transferred to new chambers in order to learn whether this will increase the recovery of isolates. Several isolates could be obtained only from material transferred through multiple diffusion chambers. This suggests that continuous cultivation in diffusion chambers adapts some microorganisms for growth under otherwise prohibitive in vitro conditions.