Experimental study on the mechanical interaction between silicon neural microprobes and rat dura mater during insertion.

In vivo insertion experiments are essential to optimize novel neural implants. Our work focuses on the interaction between intact dura mater of rats and as-fabricated single-shaft silicon microprobes realized by deep reactive ion etching. Implantation parameters like penetration force and dimpling through intact dura mater were studied as a function of insertion speed, microprobe cross-section, tip angle and animal age. To reduce tissue resistance, we proposed a unique tip sharpening technique, which was also evaluated in in vivo insertion tests. By doubling the insertion speed (between 1.2 and 10.5 mm/min), an increase of 10-35% in penetration forces was measured. When decreasing the cross-section of the microprobes, penetration forces and dimpling was reduced by as much as 30-50% at constant insertion speeds. Force was noticed to gradually decrease by decreasing tip angles. Measured penetration forces through dura mater were reduced even down to 11±3 mN compared to unsharpened (49±13 mN) probes by utilizing our unique tip sharpening technique, which is very close to exerted penetration force in the case of retracted dura (5±1.5 mN). Our findings imply that age remarkably alters the elasticity of intact dura mater. The decreasing stiffness of dura mater results in a significant rise in penetration force and decrease in dimpling. Our work is the first in vivo comparative study on microelectrode penetration through intact and retracted dura mater.

A multimodal, implantable sensor array and measurement system to investigate the suppression of focal epileptic seizure using hypothermia.

Objective.Local cooling of the brain as a therapeutic intervention is a promising alternative for patients with epilepsy who do not respond to medication.In vitroandin vivostudies have demonstrated the seizure-suppressing effect of local cooling in various animal models. In our work, focal brain cooling in a bicuculline induced epilepsy model in rats is demonstrated and evaluated using a multimodal micro-electrocorticography (microECoG) device.Approach.We designed and experimentally tested a novel polyimide-based sensor array capable of recording microECoG and temperature signals concurrently from the cortical surface of rats. The effect of cortical cooling after seizure onset was evaluated using 32 electrophysiological sites and eight temperature sensing elements covering the brain hemisphere, where injection of the epileptic drug was performed. The focal cooling of the cortex right above the injection site was accomplished using a miniaturized Peltier chip combined with a heat pipe to transfer heat. Control of cooling and collection of sensor data was provided by a custom designed Arduino based electronic board. We tested the experimental setup using an agar gel modelin vitro, and thenin vivoin Wistar rats.Main results.Spatial variation of temperature during the Peltier controlled cooling was evaluated through calibrated, on-chip platinum temperature sensors. We found that frequency of epileptic discharges was not substantially reduced by cooling the cortical surface to 30 °C, but was suppressed efficiently at temperature values around 20 °C. The multimodal array revealed that seizure-like ictal events far from the focus and not exposed to high drop in temperature can be also inhibited at an extent like the directly cooled area.Significance.Our results imply that not only the absolute drop in temperature determines the efficacy of seizure suppression, and distant cortical areas not directly cooled can be influenced.

Study of the toxic effects of an insecticide and copper sulphate on chicken embryos.

The toxic effects of the BI 58 EC insecticide (38% dimethoate) applied alone or in combination with copper sulphate were studied on chicken embryo in the early phase of development. The test materials were injected in 0.1-0.1 ml volume into the air chamber of eggs on the first day of incubation. Subsequently, on days 2 and 3 of incubation permanent preparations were made from the embryo in order to study the early developmental stage. Embryos fixed on slides and stained with osmium tetroxide solution were studied under light microscope. According to the result of the statistical evaluation, to sum up, we can say that the simultaneous administration of the test materials did not result in a significant increase in the embryo mortality, but after the combined administration the rate of embryonic mortality markedly increased. As a result of combined administrations the developmental anomalies included the apperance of a blood ring, poor development or absence of somites, the retarted development of the vascular system, the head and the body, irregular differentiation of the brain vesicles. Summarising the findings, it can be established that the insecticide treatment combined with heavy metal resulted in enhanced embryotoxicity in the case of both combinations, which was primarily manifested in an increased embryonic mortality rate.

Eye irritation study of some pesticides on chorioallantoic membrane of the egg.

The chorioallantoic membrane of chick embryo has been used extensively for many years in various fields of biological research, including virology, bacteriology and toxicology. The chorioallantoic membrane (CAM) is a complete tissue that responds to injury with a complete inflammatory reaction, this process similar to that induced by chemicals in the conjunctival tissue of the rabbit eye. A possible model for assessing the irritation potential of a chemical or product to such a vascularised tissue is the choriallantoic membrane of the embryonated hen's egg, as this is a highly vascular, thin membrane with relatively easy access for both treatment and assessment. In recent years various in vitro methods have been developed to replace the heavily criticized Draize rabbit eye test for irritation testing. One of the most studied alternative methods is the Hen's Egg Test - Chorioallantoic membrane (HET-CAM). In our studies a comparative screening was done with a set of pesticides to establish parallel data on in vitro (HET-CAM) and in vivo (Draize) results. In most cases good correlation was found between the HET-CAM assessment and results from the Draize rabbit eye test. The actual form of the HET-CAM test is a valuable pre-screen for predicting ocular irritation potential of chemicals, and can be used to reduce the number of experimental animals. The HET-CAM test is useful as a part of a battery of tests to replace the Draize rabbit eye test.

A softening laminar electrode for recording single unit activity from the rat hippocampus.

Softening neural implants that change their elastic modulus under physiological conditions are promising candidates to mitigate neuroinflammatory response due to the reduced mechanical mismatch between the artificial interface and the brain tissue. Intracortical neural probes have been used to demonstrate the viability of this material engineering approach. In our paper, we present a robust technology of softening neural microelectrode and demonstrate its recording performance in the hippocampus of rat subjects. The 5 mm long, single shank, multi-channel probes are composed of a custom thiol-ene/acrylate thermoset polymer substrate, and were micromachined by standard MEMS processes. A special packaging technique is also developed, which guarantees the stable functionality and longevity of the device, which were tested under in vitro conditions prior to animal studies. The 60 micron thick device was successfully implanted to 4.5 mm deep in the hippocampus without the aid of any insertion shuttle. Spike amplitudes of 84 µV peak-to-peak and signal-to-noise ratio of 6.24 were achieved in acute experiments. Our study demonstrates that softening neural probes may be used to investigate deep layers of the rat brain.

Effect of nanostructures on anchoring stem cell-derived neural tissue to artificial surfaces.

OBJECTIVE: Chronic application of brain implants monitoring or modulating neuronal activity are hindered by the foreign body response of the tissue. Topographical modification of implant surfaces may reduce negative tissue response by imitating the structure of the extracellular matrix and therefore affecting the attachment and behavior of neural cells. APPROACH: In our in vitro study, the effect of nanostructuring was investigated on two commercially used neural implant materials: silicon and platinum. The adhesion, survival and arrangement of neural stem cells (NE4C) and microglial cells (BV2) were investigated and compared to nanostructured and flat Si and Pt surfaces using cell viability studies and fluorescent microscopy image analysis. MAIN RESULTS: Our data indicated that neural cells established strong adhesive couplings with each other, instead of binding to the artificial surfaces. SIGNIFICANCE: The phenomena resemble some features of in vivo separation of living tissue from the implanted artificial material, providing an in vitro model for studying immune response.

Simultaneous in vivo recording of local brain temperature and electrophysiological signals with a novel neural probe.

OBJECTIVE: Temperature is an important factor for neural function both in normal and pathological states, nevertheless, simultaneous monitoring of local brain temperature and neuronal activity has not yet been undertaken. APPROACH: In our work, we propose an implantable, calibrated multimodal biosensor that facilitates the complex investigation of thermal changes in both cortical and deep brain regions, which records multiunit activity of neuronal populations in mice. The fabricated neural probe contains four electrical recording sites and a platinum temperature sensor filament integrated on the same probe shaft within a distance of 30 µm from the closest recording site. The feasibility of the simultaneous functionality is presented in in vivo studies. The probe was tested in the thalamus of anesthetized mice while manipulating the core temperature of the animals. MAIN RESULTS: We obtained multiunit and local field recordings along with measurement of local brain temperature with accuracy of 0.14 °C. Brain temperature generally followed core body temperature, but also showed superimposed fluctuations corresponding to epochs of increased local neural activity. With the application of higher currents, we increased the local temperature by several degrees without observable tissue damage between 34-39 °C. SIGNIFICANCE: The proposed multifunctional tool is envisioned to broaden our knowledge on the role of the thermal modulation of neuronal activity in both cortical and deeper brain regions.

A silicon-based microelectrode array with a microdrive for monitoring brainstem regions of freely moving rats.

OBJECTIVE: Exploring neural activity behind synchronization and time locking in brain circuits is one of the most important tasks in neuroscience. Our goal was to design and characterize a microelectrode array (MEA) system specifically for obtaining in vivo extracellular recordings from three deep-brain areas of freely moving rats, simultaneously. The target areas, the deep mesencephalic reticular-, pedunculopontine tegmental-and pontine reticular nuclei are related to the regulation of sleep-wake cycles. APPROACH: The three targeted nuclei are collinear, therefore a single-shank MEA was designed in order to contact them. The silicon-based device was equipped with 3 × 4 recording sites, located according to the geometry of the brain regions. Furthermore, a microdrive was developed to allow fine actuation and post-implantation relocation of the probe. The probe was attached to a rigid printed circuit board, which was fastened to the microdrive. A flexible cable was designed in order to provide not only electronic connection between the probe and the amplifier system, but sufficient freedom for the movements of the probe as well. MAIN RESULTS: The microdrive was stable enough to allow precise electrode targeting into the tissue via a single track. The microelectrodes on the probe were suitable for recording neural activity from the three targeted brainstem areas. SIGNIFICANCE: The system offers a robust solution to provide long-term interface between an array of precisely defined microelectrodes and deep-brain areas of a behaving rodent. The microdrive allowed us to fine-tune the probe location and easily scan through the regions of interest.

Teratogenicity study of some pesticides in chicken embryos.

The use of pesticides involves the risk of poisoning on wild animals. Teratological tests carried out on avian embryos provide useful data for environmental protection and facilitate the development of environment-friendly chemical plant protection techniques. A 30% dimethoate containing insecticide formulation (BI 58 EC) and a 20% benfluralin containing herbicide formulation (Flubalex) and a 960 g/l S-metolachlor containing herbicide formulation (Dual Gold 960 EC) were studied in chicken embryos after single administration by immersion and injection technique. Treatment was done on day 0 of incubation. Applied concentration of pesticides were 0.1% (dimethoate) and 2.05% (S-metolachlor) and 0.375% (benfluralin) corresponding to that used in plant protection practice. Test materials were injected into the air chamber in a volume of 0.1 ml/egg, or eggs were treated by the immersion technique for 30 min. at 37 degrees C. Evaluation was done on day 19 of incubation. Injection treatment: the administration of S-metolachlor and benfluralin did not result a significant decrease in the average body weight of embryos. At the same time the body weight of embryos significantly decreased because of single administration of dimethoate. The embryomortality increased markedly after the administration of test materials (S-metolachlor, benfluralin, dimethoate). Immersion treatment: the administration of S-metolachlor and benfluralin and dimethoate did not result a significant decrease in the average body weight of embryos. The rate of embryomortality was low after the administration of S-metolachlor, benfluralin and dimethoate. After the immersion and the injection treatment the incidences of developmental anomalies were sporadic. In summary it can be established that the injection treatment was more toxic than immersion technique of the test materials in our study.

Irritative effects of some pesticides and a technical component on tissue structure of the chorioallantoic membrane.

The chorioallantoic membrane (CAM) is a complete tissue that responds to injury with a complete inflammatory reaction, this process similar to that induced by chemicals in the conjunctival tissue of the rabbit eye. During the study chemicals are placed directly onto the chorioallantoic membrane and the occurrence of vascular injury or coagulation in response to a compound is as an indication of the potential of a chemical to damage mucous membranes. In our study irritant pesticides (Fusilade S, Karathane LC) and a technical pesticide component (Trend) were tested and their effects on the tissue structures of CAM were examined. After treatment with the test materials, first lysis and then haemorrhage were observed macroscopically on CAM. In histological pictures stained with H-E the rupture of the blood vessel wall was seen and blood was observed around the blood vessels in the middle layer. The histological findings correlated well with the macroscopic appearance in this study. In general a good correlation was found between the HET-CAM results and reported data from Draize test. The subjective nature of the evaluation is reduced through the histological examination of treated CAM. The HET-CAM test can be a useful component of a battery of tests needed for replacing the Draize rabbit eye irritation test.

Toxicity of a dichlorvos containing insecticide formulation and an atrazine containing herbicide formulation in chicken embryos after individual administration.

A 50% dichlorvos containing insecticide formulation (Unifosz 50 EC) and a 50% atrazine containing herbicide formulation (Hungazin PK 50 WP) were studied in chicken embryos after administration as single compounds. Applied concentrations of dichlorvos were 0.1% (corresponding to the plant protection practice), 0.05%, 0.02%, 0.01%. Applied concentrations of atrazine were 0.66% (corresponding to the plant protection practice), 0.33%, 0.132%, 0.066%. The test materials were injected directly into the air-chamber of eggs on day 0 of the hatching period and evaulation was carried out on day 19 of incubation. The chicken embryos were examined for the following: rate of embryo mortality, body mass, type of developmental anomalies. After the single administrations of dichlorvos containing insecticide formulation and atrazine containing herbicide formulation on day 0 of incubation, the average body weight of chicken embryos significantly did not decrease as compared to the control. After the individual administrations of pesticides the incidence of developmental anomalies was sporadic. The embryonic mortality markedly increased at the highest concentrations of pesticides. The rate of embrio mortality were 61% (dichlorvos insecticide containing formulation) and 52% (atrazine containing herbicide formulation). In summary, the 50% dichlorvos containing insecide formulation (Unifosz 50 EC) and the 50% atrazine containing herbicide formulation (Hungazin PK 50 WP) were toxic to the developing chicken embryos at the highest concentration in our study. The toxic effect was expressed in the high rate of embrio mortality.

Testing rocuronium-induced neuromuscular blockade at the stapedius muscle using stapedius reflex measurements.

BACKGROUND: Neuromuscular monitoring prior to emergence from anaesthesia has been shown to be necessary to achieve adequate airway protection in order to decrease postoperative pulmonary complications. In the present study we hypothesized that stapedius reflex measurement allows the detection of residual neuromuscular blockade using the stapedius muscle following the administration of rocuronium. PATIENTS AND METHODS: Parallel stapedius and acceleromyographic measurements were performed on 20 patients undergoing cholecystectomy. Acceleromyographic measurements were continuously performed during the course of anaesthesia, whereas the stapedius reflex was measured on different occasions: (1) after premedication but before anaesthesia induction, (2) after induction, but before administration of muscle relaxant, (3) after administration of muscle relaxant, (4) during the course of surgical anaesthesia at regular intervals, and (5) continuously performed during emergence from anaesthesia, until the stapedius reflex threshold returned to normal. RESULTS: The intensity of the sound energy at which the stapedius reflex is detectable was similar: 89.5 ± 9.9 dB(mean ± SD) after premedication and after anaesthetic induction. However, after administration of rocuronium, when the twitch height decreased to 5%, the stapedius reflex disappeared, indicating a total block of the stapedius muscle.During the recovery phase (twitch>10%) significantly higher sound energies compared to baseline values were necessary to evoke the reflex, indicating residual inhibition of the stapedius muscle. At the point where stapedius reflex threshold returned to normal the twitch height averaged about 50% showing low sensitivity of the tympanometry in detecting residual neuromuscular blockade. CONCLUSIONS: The neuromuscular effect of rocuronium on the stapedius muscle can be detected using stapedius reflex measurements. Due to its methodological limitation and low sensitivity, the method cannot be recommended for the monitoring of residual neuromuscular blockade.