Impact of degradable nanowires on long-term brain tissue responses.

BACKGROUND: A promising approach to improve the performance of neural implants consists of adding nanomaterials, such as nanowires, to the surface of the implant. Nanostructured interfaces could improve the integration and communication stability, partly through the reduction of the cell-to-electrode distance. However, the safety issues of implanted nanowires in the brain need to be evaluated and understood before nanowires can be used on the surface of implants for long periods of time. To this end we here investigate whether implanted degradable nanowires offer any advantage over non-degradable nanowires in a long-term in vivo study (1 year) with respect to brain tissue responses. RESULTS: The tissue response after injection of degradable silicon oxide (SiOx)-coated gallium phosphide nanowires and biostable hafnium oxide-coated GaP nanowires into the rat striatum was compared. One year after nanowire injection, no significant difference in microglial or astrocytic response, as measured by staining for ED1 and glial fibrillary acidic protein, respectively, or in neuronal density, as measured by staining for NeuN, was found between degradable and biostable nanowires. Of the cells investigated, only microglia cells had engulfed the nanowires. The SiOx-coated nanowire residues were primarily seen in aggregated hypertrophic ED1-positive cells, possibly microglial cells that have fused to create multinucleated giant cells. Occasionally, degradable nanowires with an apparently intact shape were found inside single, small ED1-positive cells. The biostable nanowires were found intact in microglia cells of both phenotypes described. CONCLUSION: The present study shows that the degradable nanowires remain at least partly in the brain over long time periods, i.e. 1 year; however, no obvious bio-safety issues for this degradable nanomaterial could be detected.

Psychometric evaluation of the Dundee Ready Educational Environment Measure: Swedish version.

BACKGROUND: The Dundee Ready Educational Environment Measure (DREEM) has been used in various studies to evaluate the educational environment. However, psychometric evaluations of the instrument seem sparse, for all known versions of the instrument. AIM: The aim was to psychometrically evaluate the Swedish version of the DREEM instrument. METHOD: A total of 503 students (undergraduate medicine), aged 19-46 years, in semesters 2, 6 and 10 were included in the study. Validity was evaluated through analysis of construct validity and reliability. RESULTS: The instrument had in general both acceptable validity and reliability. Due to a rather poor model fit in the confirmatory factor analysis, an explorative factor analysis was also employed which suggested a new five-factor solution for the instrument. CONCLUSIONS: The Swedish version of the DREEM instrument is shown to be valid and reliable, except for the factor structure. The new five-factor solution found in this study is not proven to be a superior measurement model compared with the original, but could be seen as an alternative model to the original, where the strong and weak areas are somewhat more easily identified.

[Portfolio - a tool for making learning and competence development visible]. / Portföljen synliggör lärandet och kompetensutvecklingen.

Portfolio used in education can be defined as a collection of documentation of performed learning activities, feedback, and progress. Currently, the documentation is electronic, hence the term e-portfolio is used. The portfolio must have a clear purpose and be aligned with the learning outcomes of the program. To be successfully implemented a portfolio must be an integral part of the education with defined tasks for both the students and the teachers. Students and teacher support in how to use the portfolio is essential especially in the beginning of the program. Learning analytics enables teachers to identify and develop support for students at risk of not achieving the outcomes.

UVB irradiation induces contralateral changes in galanin, substance P and c-fos immunoreactivity in rat dorsal root ganglia, dorsal horn and lateral spinal nucleus.

The selection of control group is crucial, as the use of an inadequate group may strongly affect the results. In this study we examine the effect on contralateral tissue protein levels, in a model of unilateral UVB irradiation, as the contralateral side is commonly used as a control. Previous studies have shown that UVB irradiation increases immunoreactivity for inflammatory regulated neuropeptides. Unilateral UVB irradiation of rat hind paw was performed and corresponding contralateral spinal cord and dorsal root ganglia (DRG) were collected 2-96 h after and investigated for changes in galanin, substance P and c-fos immunoreactivity. Control tissue was collected from naïve rats. Measurement of skin blood flow from contralateral heel hind paws (Doppler), revealed no change compared to naïve rats. However, UVB irradiation caused a significant reduction in the contralateral proportion of galanin immunopositive DRG neurons, at all-time points, as well as an increase in the contralateral spinal cord dorsal horn, around the central canal and in the lateral spinal nucleus (2-48 h). The contralateral proportion of SP positive DRG neurons and dorsal horn immunoreactivity was unchanged, whereas the lateral spinal nucleus area showed increased immunoreactivity (48 h). UVB irradiation also induced a slight contralateral upregulation of c-fos in the dorsal horn/central canal area (24 and 48 h). In summary, unilateral UVB irradiation induced contralateral changes in inflammatory/nociceptive neuropeptides in spinal cord and afferent pathways involved in pain signaling already within 24 h, a time point when also ipsilateral neurochemical/physiological changes have been reported for rats and humans.

Comparing the educational environment (as measured by DREEM) at two different stages of curriculum reform.

BACKGROUND: The medical programme at Lund University, Sweden, has undergone curricular reform over several stages, which is still ongoing. Students have been somewhat negative in their evaluations of the education during this time. AIM: To find out how the students perceived the educational climate using the Dundee Ready Education Environment Measure (DREEM), and to compare the findings taken at two given points in time. METHOD: The DREEM instrument was distributed in semesters 2, 6 and 10 in 2003 and 2005, to a total of 503 students. RESULTS: The students rated their climate as positive. The total DREEM score (145) was somewhat higher than other published results and in the same range as for other reformed curricula. There was hardly any difference between the genders in their perceptions of the climate. Certain items were rated low and became subject of development between the measurements. These items concerned a perceived lack of a support system for stressed students and a lack of feedback and constructive criticism from teachers. Some improvement was detected in 2005. CONCLUSION: The educational climate was high in a reformed curriculum and could be maintained high during on-going curricular reform. Educational development resulted in better results on some items.

Nanowire biocompatibility in the brain--looking for a needle in a 3D stack.

We investigated the brain-tissue response to nanowire implantations in the rat striatum after 1, 6, and 12 weeks using immunohistochemistry. The nanowires could be visualized in the scar by confocal microscopy (through the scattered laser light). For the nanowire-implanted animals, there is a significant astrocyte response at week 1 compared to controls. The nanowires are phagocytized by ED1 positive microglia, and some of them are degraded and/or transported away from the brain.

UVB irradiation induces rapid changes in galanin, substance P and c-fos immunoreactivity in rat dorsal root ganglia and spinal cord.

Recent studies have shown that UVB irradiation induces primary and secondary hyperalgesia in rats and humans peaking about 24h after UVB exposure. In the present study we investigated the changes in galanin, substance P and c-fos immunoreactivity in rat DRG and spinal cord at the L5 level 2-96h after UVB irradiation. UVB irradiation of the heel area in rats almost increased the skin blood flow two-fold 24h after irradiation as measured by laser Doppler technique. UVB irradiation induced a significant reduction of the proportion of galanin positive DRG neurons for all time points, except at 12h. In the spinal cord, UVB irradiation induced increased immunoreactivity for galanin in the dorsal horn, the area around the central canal and interestingly also in the lateral spinal nucleus 12-96h after exposure. For substance P the proportion of substance P positive neurons was unchanged but UVB irradiation induced increased substance P immunoreactivity in the dorsal part of the spinal cord 48h after irradiation. UVB irradiation also induced c-fos immunoreactivity in the dorsal horn and the area around the central canal 24 and 48h after exposure. This translational model of UVB irradiation will induce rapid changes of neuropeptides implicated in nociceptive signaling in areas known to be of importance for nociception in a time frame, about 24h after exposure, where also neurophysiological alteration have been described in humans and rats.

Axonal outgrowth on nano-imprinted patterns.

Nanotechnology has provided methods to fabricate surface patterns with features down to a few nm. If cells or cell processes exhibit contact guidance in response to such small patterns is an interesting question and could be pertinent for many applications. In the present study we investigated if axonal outgrowth was affected by nano-printed patterns in polymethylmethacrylate (PMMA)-covered silicon chips. To this end adult mouse sympathetic and sensory ganglia were mounted in Matrigel on the chips close to the nano-patterns. The patterns consisted of parallel grooves with depths of 300 nm and varying widths of 100-400 nm. The distance between two adjacent grooves was 100-1600 nm. The chips were cultured in medium containing 25 ng/ml of nerve growth factor to stimulate axonal outgrowth. After 1 week of incubation, axonal outgrowth was investigated by immunocytochemistry or scanning electron microscopy. Axons displayed contact guidance on all patterns. Furthermore, we found that the nerve cell processes preferred to grow on ridge edges and elevations in the patterns rather than in grooves, a seemingly claustrophobic behavior. We conclude that axons of peripheral neurons might be guided by nanopatterns on PMMA when the lateral features are 100 nm or larger. The present results can be utilized for nerve regenerating scaffolds or the construction of a stable, high-resolution electronic interface to neurons, which is required for future brain machine interfaces.

Altered behavioural responses and functional recovery in rats following sciatic nerve compression and early vs late decompression.

OBJECTIVE: The aim of the study was to examine sensory behaviour and functional recovery in rats during nerve compression and after decompression. Compression injury is a far more common condition than nerve transection. The condition is characterised by numbness and a tingling/burning sensation, and some patients experience pain and allodynia during compression or after decompression treatment. The aetiology is in many cases unknown. Thus, further studies are of great importance for the understanding of this condition. METHODS: In the present study, behavioural responses to tactile stimulation, thermal pain, as well as functional sensorimotor behaviour were investigated in rats before, during severe compression, and after decompression. The sciatic nerve of the rats was experimentally compressed for 3 or 28 days, whereafter surgical release, i.e. decompression, of the nerve was performed and the rats were examined up to ∼9 weeks. RESULTS: An altered behaviour was found in response to compression injury, which is mitigated after early decompression treatment. CONCLUSIONS: These findings indicate that early intervention during severe compression injuries is of great importance for recovery and restoration of nerve function and, thus, should have an impact on clinical routines regarding treatment of compression injuries.

Size-dependent long-term tissue response to biostable nanowires in the brain.

Nanostructured neural interfaces, comprising nanotubes or nanowires, have the potential to overcome the present hurdles of achieving stable communication with neuronal networks for long periods of time. This would have a strong impact on brain research. However, little information is available on the brain response to implanted high-aspect-ratio nanoparticles, which share morphological similarities with asbestos fibres. Here, we investigated the glial response and neuronal loss in the rat brain after implantation of biostable and structurally controlled nanowires of different lengths for a period up to one year post-surgery. Our results show that, as for lung and abdominal tissue, the brain is subject to a sustained, local inflammation when biostable and high-aspect-ratio nanoparticles of 5 µm or longer are present in the brain tissue. In addition, a significant loss of neurons was observed adjacent to the 10 µm nanowires after one year. Notably, the inflammatory response was restricted to a narrow zone around the nanowires and did not escalate between 12 weeks and one year. Furthermore, 2 µm nanowires did not cause significant inflammatory response nor significant loss of neurons nearby. The present results provide key information for the design of future neural implants based on nanomaterials.

PACAP mRNA is expressed in rat spinal cord neurons.

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.

Expression of orphanin FQ/nociceptin and its receptor in rat peripheral ganglia and spinal cord.

Expression of the neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, the opioid receptor-like receptor (ORL1), have been found to have a wide distribution in the central nervous system, and in brain areas involved in sensory perception in particular. The effects of OFQ/N on, e.g., sensory transmission are very complex, and a modulatory effect on pain perception has been suggested. We therefore wanted to investigate the distribution of OFQ/N and ORL1 in the spinal cord and DRG, and also in SCG and some other peripheral tissues. The methods used were in situ hybridization, immunohistochemistry and ligand binding. We found that OFQ/N and ORL1 mRNA are expressed in DRG; primarily in small and large neurons, respectively. In spinal cord, mRNA for OFQ/N and ORL1 is expressed in neurons in laminae I, II and X, and in ventral horn neurons. Further, immunoreactivity for OFQ/N is observed in fibers and neurons in the superficial laminae of the dorsal horn and around the central canal, and also in neurons in the ventral horn of the spinal cord. Receptor ligand binding to the spinal cord grey matter is demonstrated, primarily concentrated to the dorsal horn and around the central canal, and also to medium and large size DRG neurons. These findings on the morphological distribution pattern of OFQ/N and ORL1 at the cellular level may support the notion that OFQ/N is involved in modulating pain transmission. Further, expression of OFQ/N and ORL1 mRNA was also found in SCG, whereas expression was undetectable in skin.

Tissue reactions evoked by porous and plane surfaces made out of silicon and titanium.

Square-shaped silicon or titanium implants with plane or porous surfaces surrounded by a rim of silicone were implanted in the rat abdominal wall for evaluation of the tissue response after one, six, or 12 weeks. Cell damage was identified as increased membrane permeability using fluorescence microscopy by injection of propidium iodide prior to the killing of the rats. Capsule thickness and immunohistochemical quantification of macrophages were used as a further measure of the foreign-body reaction. There were no significant differences in capsular cell densities for macrophages, total cells (macrophages, fibroblasts, and other cells), or necrotic cells at the different time points for the four surfaces studied. However, significant differences in the kinetics of the response were found between plane surfaces compared with porous ones. Both types of plane surfaces developed a significant increase in capsule thickness over time in contrast to the porous implants. Porous silicon displayed a significant decrease in total cells in the reactive capsule over time. Furthermore, porous silicon and titanium surfaces displayed a significant decrease in total cell numbers at the implant interface between six and 12 weeks. The present study demonstrated that implanted silicon elicited soft-tissue reactions comparable to that of titanium.

Locally-applied collagenase and regeneration of transsected and repaired rat sciatic nerves.

The possible effects of collagenase on peripheral nerve regeneration were evaluated after epineurial repair of rat sciatic nerves. In the control group the repair site was covered by fibrin adhesive and infused with isotonic saline and in the experimental group collagenase was infused into the fibrin adhesive. In the short term study the regeneration distance was measured by a pinch test four, six, or eight days postoperatively. In the long term study the evaluation of nerve regeneration and recovery of motor function was made by testing the tetanic contraction force of the anterior tibial muscle three months postoperatively. There were no significant differences between the two groups in either the short or long term. We conclude that locally-applied collagenase had no effect on peripheral nerve regeneration.

A comparison of peripheral nerve regeneration in acellular muscle and nerve autografts.

Regeneration of the rat sciatic nerve through acellular muscle and nerve autografts was evaluated 6-28 days postoperatively by the sensory pinch test, immunocytochemical staining for neurofilaments, and light and electron microscopy. Data points generated by the pinch test were plotted against postoperative time periods and by the use of regression analysis the initial delay period for muscle grafts was determined to 10.3 days. This value was similar to that previously published for acellular nerve grafts (9.5 days), but significantly longer than that for fresh nerve grafts (3.6 days). The calculated regeneration rate (slope of the regression line) for muscle grafts (1.8 mm/day) did not differ significantly (p > 0.05) from that calculated for acellular nerve grafts (2.1 mm/day) or for fresh nerve grafts (1.5 mm/day). The front of regenerating axons shown by axonal neurofilament staining confirmed the pinch test results. Both types of acellular grafts were repopulated with host non-neuronal cells and the muscle graft contained occasional ectopic muscle fibres. Remnants of graft basal laminae were evident at the ultrastructural level. These results indicate the suitability of either acellular muscle or nerve grafts for nerve repair despite their prolonged initial delay periods compared with conventional fresh nerve grafts.

Multiple implants do not aggravate the tissue reaction in rat brain.

Chronically implanted microelectrodes are an invaluable tool for neuroscientific research, allowing long term recordings in awake and behaving animals. It is known that all such electrodes will evoke a tissue reaction affected by its' size, shape, surface structure, fixation mode and implantation method. However, the possible correlation between tissue reactions and the number of implanted electrodes is not clear. We implanted multiple wire bundles into the brain of rats and studied the correlation between the astrocytic and microglial reaction and the positioning of the electrode in relation to surrounding electrodes. We found that an electrode implanted in the middle of a row of implants is surrounded by a significantly smaller astrocytic scar than single ones. This possible interaction was only seen between implants within the same hemisphere, no interaction with the contralateral hemisphere was found. More importantly, we found no aggravation of tissue reactions as a result of a larger number of implants. These results highlight the possibility of implanting multiple electrodes without aggravating the glial scar surrounding each implant.

Can histology solve the riddle of the nonfunctioning electrode? Factors influencing the biocompatibility of brain machine interfaces.

Neural interfaces hold great promise to become invaluable clinical and diagnostic tools in the near future. However, the biocompatibility and the long-term stability of the implanted interfaces are far from optimized. There are several factors that need to be addressed and standardized when improving the long-term success of an implanted electrode. We have chosen to focus on three key factors when evaluating the evoked tissue responses after electrode implantation into the brain: implant size, fixation mode, and evaluation period. Further, we show results from an ultrathin multichannel wire electrode that has been implanted in the rat cerebral cortex for 1 year. To improve biocompatibility of implanted electrodes, we would like to suggest that free-floating, very small, flexible, and, in time, wireless electrodes would elicit a diminished cell encapsulation. We would also like to suggest standardized methods for the electrode design, the electrode implantation method, and the analyses of cell reactions after implantation into the CNS in order to improve the long-term success of implanted neural interfaces.

Implant size and fixation mode strongly influence tissue reactions in the CNS.

The function of chronic brain machine interfaces depends on stable electrical contact between neurons and electrodes. A key step in the development of interfaces is therefore to identify implant configurations that minimize adverse long-term tissue reactions. To this end, we here characterized the separate and combined effects of implant size and fixation mode at 6 and 12 weeks post implantation in rat (n = 24) cerebral cortex. Neurons and activated microglia and astrocytes were visualized using NeuN, ED1 and GFAP immunofluorescence microscopy, respectively. The contributions of individual experimental variables to the tissue response were quantified. Implants tethered to the skull caused larger tissue reactions than un-tethered implants. Small diameter (50 µm) implants elicited smaller tissue reactions and resulted in the survival of larger numbers of neurons than did large diameter (200 µm) implants. In addition, tethering resulted in an oval-shaped cavity, with a cross-section area larger than that of the implant itself, and in marked changes in morphology and organization of neurons in the region closest to the tissue interface. Most importantly, for implants that were both large diameter and tethered, glia activation was still ongoing 12 weeks after implantation, as indicated by an increase in GFAP staining between week 6 and 12, while this pattern was not observed for un-tethered, small diameter implants. Our findings therefore clearly indicate that the combined small diameter, un-tethered implants cause the smallest tissue reactions.

Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons.

Identification of the molecule(s) that globally induce a robust regenerative state in sensory neurons following peripheral nerve injury remains elusive. A potential candidate is brain-derived neurotrophic factor (BDNF), the sole neurotrophin upregulated in sensory neurons after peripheral nerve injury. Here we tested the hypothesis that BDNF plays a critical role in the regenerative response of mature rat sensory neurons following peripheral nerve lesion. Neutralization of endogenous BDNF was performed by infusing BDNF antibodies intrathecally via a mini-osmotic pump for 3 days at the level of the fifth lumbar dorsal root ganglion, immediately following unilateral spinal nerve injury. This resulted in decreased expression of the injury/regeneration-associated genes growth-associated protein-43 and Talpha1 tubulin in the injured sensory neurons as compared to injury plus control IgG infused or injury alone animals. Similar results were observed following inhibition of BDNF expression by intrathecal delivery of small interfering RNAs (siRNA) targeting BDNF starting 3 days prior to injury. The reduced injury/regeneration-associated gene expression correlated with a significantly reduced intrinsic capacity of these neurons to extend neurites when assayed in vitro. In contrast, delayed infusion of BDNF antibody for 3 days beginning 1 week post-lesion had no discernible influence on the elevated expression of these regeneration-associated markers. These results support an important role for endogenous BDNF in induction of the cell body response in injured sensory neurons and their intrinsic ability to extend neurites, but BDNF does not appear to be necessary for maintaining the response once it is induced.

Porous silicon as a potential electrode material in a nerve repair setting: Tissue reactions.

We compared porous silicon (pSi) with smooth Si as chip-implant surfaces in a nerve regeneration setting. Silicon chips can be used for recording neural activity and are potential nerve interface devices. A silicon chip with one smooth and one porous side inserted into a tube was used to bridge a 5 mm defect in rat sciatic nerve. Six or 12 weeks later, new nerve structures surrounded by a perineurium-like capsule had formed on each side of the chip. The number of regenerated nerve fibers did not differ on either side of the chip as shown by immunostaining for neurofilaments. However, the capsule that had formed in contact with the chip was significantly thinner on the porous side than on the smooth side. Cellular protrusions had formed on the pSi side and the regenerated nerve tissue was found to attach firmly to this surface, while the tissue was hardly attached to the smooth silicon surface. We conclude that a pSi surface, due to its large surface area, diminished inflammatory response and firm adhesion to the tissue, should be a good material for the development of new implantable electronic nerve devices.