A critical review of cell culture strategies for modelling intracortical brain implant material reactions.

The capacity to predict in vivo responses to medical devices in humans currently relies greatly on implantation in animal models. Researchers have been striving to develop in vitro techniques that can overcome the limitations associated with in vivo approaches. This review focuses on a critical analysis of the major in vitro strategies being utilized in laboratories around the world to improve understanding of the biological performance of intracortical, brain-implanted microdevices. Of particular interest to the current review are in vitro models for studying cell responses to penetrating intracortical devices and their materials, such as electrode arrays used for brain computer interface (BCI) and deep brain stimulation electrode probes implanted through the cortex. A background on the neural interface challenge is presented, followed by discussion of relevant in vitro culture strategies and their advantages and disadvantages. Future development of 2D culture models that exhibit developmental changes capable of mimicking normal, postnatal development will form the basis for more complex accurate predictive models in the future. Although not within the scope of this review, innovations in 3D scaffold technologies and microfluidic constructs will further improve the utility of in vitro approaches.

Cytotoxic activity of extracts from Hypochaeris radicata.

Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) and control, unstressed (UE) plants were incubated with myelinating spinal cord cultures (MSCC) established from fetal Swiss mice, and with spinal ganglion cultures (SGC) and dermal fibroblast cultures derived from neonatal mouse tissues. Cytotoxicity in culture monolayers was evaluated both morphologically by microscopy and by release of lactate dehydrogenase activity into culture supernates. Three different SGC preparations were exposed to a single H. radicata extract and single preparations of fibroblasts and MSCC were exposed to three different extracts. Repin, a plant-derived sesquiterpene lactone neurotoxin, was included as a positive control. Significant dose-dependent cytotoxicity was seen within 24 h in all three culture types when incubated with SE or repin. Complete morphologic destruction of culture monolayers was induced by the highest concentrations tested of SE (100 µg/mL) and repin (30 µg/mL). Cytotoxic effect of SE was significantly greater than that of UE for all three cell types and was not due to copper contamination of the extract. This study has identified a cytotoxic activity in leaf exudates of H. radicata that was upregulated by the model stressor, copper chloride.

Functional outcomes following ceramic total joint replacement for hallux rigidus.

INTRODUCTION: The aim of this study was to evaluate functional outcomes following ceramic arthroplasty used in the treatment of osteoarthritis of the hallux metatarsophalangeal (MTP) joint. MATERIALS AND METHODS: Thirty-seven consecutive patients who underwent press-fit ceramic joint arthroplasty were identified. Joint movement, gait pressure studies, radiographs, patient's outcome based on the American Orthopaedic Foot and Ankle Society (AOFAS) scale, hallux metatarsal phalangeal-interphalangeal index (HMPI) and visual analogue pain scales were assessed. RESULTS: Mean follow-up was 33 (12-60) months. Ninety-two percent of patients were satisfied with the surgery. AOFAS and HMPI scores were good to excellent in more than 90%. Six implants had lucent lines of greater than 2mm at 18 months. Three of these joints also had subsidence of both components. There was no correlation between implant loosening and patient outcomes. Mean hallux pressure at toe-off decreased from 7.1 to 3.5 N cm⁻² (p<0.01) equalising with normal contralateral toe pressure. Three patients required revision surgery and one patient had a transient wound infection. CONCLUSIONS: Good to excellent results have been achieved following ceramic total MTP joint arthroplasty. The clinical relevance of progressive lucencies around the implant is uncertain and longer follow-up may identify subsidence and ultimate failure.

Myelinated, synapsing cultures of murine spinal cord--validation as an in vitro model of the central nervous system.

Research in central nervous system (CNS) biology and pathology requires in vitro models, which, to recapitulate the CNS in vivo, must have extensive myelin and synapse formation under serum-free (defined) conditions. However, finding such a model has proven difficult. The technique described here produces dense cultures of myelinated axons, with abundant synapses and nodes of Ranvier, that are suitable for both morphological and biochemical analysis. Cellular and molecular events were easily visualised using conventional microscopy. Ultrastructurally, myelin sheaths were of the appropriate thickness relative to axonal diameter (G-ratio). Production of myelinated axons in these cultures was consistent and repeatable, as shown by statistical analysis of multiple experimental repeats. Myelinated axons were so abundant that from one litter of embryonic mice, myelin was produced in amounts sufficient for bulk biochemical analysis. This culture method was assessed for its ability to generate an in vitro model of the CNS that could be used for both neurobiological and neuropathological research. Myelin protein kinetics were investigated using a myelin fraction isolated from the cultures. This fraction was found to be superior, quantitatively and qualitatively, to the fraction recovered from standard cultures of dissociated oligodendrocytes, or from brain slices. The model was also used to investigate the roles of specific molecules in the pathogenesis of inflammatory CNS diseases. Using the defined conditions offered by this culture system, dose-specific, inhibitory effects of inflammatory cytokines on myelin formation were demonstrated, unequivocally. The method is technically quick, easy and reliable, and should have wide application to CNS research.

In-shoe pressure distribution in "unstable" (MBT) shoes and flat-bottomed training shoes: a comparative study.

BACKGROUND: Footwear comfort in many clinical situations is dependent on the ability of the 'shoe' to redistribute plantar pressure. Offloading the metatarsal heads may be achieved by fitting an insole, but recently a new design of shoe with a curved under sole (Masai Barefoot Technology or "MBT shoe") has been advocated. The aim of this study was to directly assess the effect of such shoes on gait pattern. METHODS: Normal subjects were recruited and asked to walk sequentially in (a) flat-soled training shoes and (b) midfoot bearing shoes (MBT shoe). Mean and peak pressures in four anatomically defined areas of the foot, and the total area of sole contact were measured electronically by an in-shoe system (Pedar Ltd., UK). PRINCIPAL RESULTS: Standing in the Masai shoes resulted in a 21% lesser peak pressure under the midfoot and an 11% lesser peak pressure under the heel in comparison to the figures found when patients wore their training shoes. There was a 76% compensatory increase in pressure under the toes. In essence there was a significant shift in pressure towards the front of the foot.

Honey dressing versus paraffin tulle gras following toenail surgery.

OBJECTIVE: Anecdotal reports suggest that certain honey dressings have a positive effect on wound healing. However, there is limited empirical evidence supporting its use. This double-blind randomised controlled trial investigated the effect of a honey dressing on wound healing following toenail surgery with matrix phenolisation. METHOD: Participants (n=100) were randomly assigned to receive either an active manuka honey dressing (n=52) or paraffin-impregnated tulle gras (n=48). The primary outcome was time (days) taken for complete re-epithelialisation of the nail bed. RESULTS: Mean healing times were 40.30 days (SD 18.21) for the honey group and 39.98 days (SD 25.42) for the paraffin tulle gras group. Partial avulsion wounds healed statistically significantly faster (p=0.01) with paraffin tulle gras (19.62 days, SD 9.31) than with the honey dressing (31.76 days, SD 18.8), but no significant difference (p=0.21) was found following total avulsion when comparing honey (45.28 days, SD 18.03.) with paraffin tulle gras dressings (52.03 days, SD 21.3). CONCLUSION: The results suggest that patients may benefit more from paraffin tulle gras dressings than honey dressings following partial toenail avulsion. No statistically significant difference was found for healing times after total toenail avulsion, although the marginal benefit of the honey dressing on these healing times warrants further investigation.

Amylin induces natriuresis by a central angiotensin-dependent mechanism.

This study provides evidence that amylin acts centrally to increase sodium excretion in the sheep. Amylin was infused at 8 mg/h into a carotid artery (IC), via a lateral ventricle (ICV), intravenously (IV) or intra-renally (IR) into conscious sheep (n=5 per group). Renal sodium excretion increased by at least 3-fold after 1 h of amylin infusion by ICV (66+/-14 to 367+/-35 mmol/min) and IC (78+/-14 to 244+/-22 mmol/min) routes of administration. Amylin infusion IV caused a 1.5-fold increase in sodium excretion while IR infusion did not have a significant effect. The natriuretic effect of ICV infused amylin was blocked by pre-treatment with the angiotensin AT1 receptor antagonist, losartan (1 mg/h). No changes in blood pressure or heart rate were recorded at this dose of amylin by any route of administration. Plasma renin concentration increased (1.32+/-0.22 to 2.55+/-0.73 pmol/Ang I/h; P<0.05) following IR infusion of amylin, and remained unchanged when amylin was infused by the other routes of administration. We conclude that amylin causes changes in sodium excretion in sheep through a central, angiotensin-dependent pathway and that amylin may increase renin secretion by a direct effect on the kidney.

Plp gene regulation in the developing murine optic nerve: correlation with oligodendroglial process alignment along the axons.

The factors regulating the expression and splicing of the major myelin gene, proteolipid protein (Plp), are unclear. The gene encodes two splice variants, Plp and Dm20. During active myelination, transcription of the Plp gene is markedly upregulated and the splice variant ratio becomes Plp-mRNA dominant. We hypothesised that these aspects of Plp gene regulation are linked to overt axonal contact. Using the developing optic nerve of mice, we demonstrate that alignment of oligodendroglial processes with the axon correlates with both the expression of Plp-mRNA and the transcriptional upregulation of the gene. We test the above hypothesis more extensively in a subsequent study.

Dysplasia of the caudal vertebral articular facets in four dogs: results of radiographic, myelographic and magnetic resonance imaging investigations.

Congenital anomalies of the vertebral column associated with aberrations of one of the primary vertebral ossification centres have been frequently described in the veterinary literature, but clinically significant abnormalities of secondary vertebral ossification centres, particularly involving the caudal articular processes, are much less frequently reported. This paper describes three dogs with aplasia and one dog with hypoplasia of the caudal vertebral articular processes. Thoracolumbar spinal cord compression and ataxia was evident in the three dogs with aplasia but no clinical signs were evident in the dog with hypoplasia. The radiographic appearance was similar in all four cases, with aplasia or hypoplasia of the caudal articular facets at one or more intervertebral joints in the thoracolumbar region. Bone proliferation was evident secondary to an associated degenerative joint disease. Compensatory hyperplasia of the adjacent cranial articular facets and ligamentum flavum protruded into the vertebral canal, resulting in a compressive myelopathy observed by myelography and magnetic resonance imaging.

Short report: encouraging GPs to complete postal questionnaires--one big prize or many small prizes? A randomized controlled trial.

BACKGROUND: Low response rates to surveys are a problem in general practice. There is evidence that offering GPs incentives improves response rates to postal questionnaires. However, there is less evidence about the most effective form of incentive. OBJECTIVE: Our trial aimed to maximize response to a postal questionnaire and to test the most effective form of incentive. METHODS: The study involved a randomized controlled trial of a postal survey RESULTS: The incentive of a lottery for six bottles of champagne generated a response rate of 79%. Furthermore, one chance of six bottles generated 9% more responses than six chances of one bottle. CONCLUSIONS: This study has established that, among incentives for postal questionnaires, one big prize improves the yield more than many small prizes despite the lower odds of winning. It has also confirmed that offering a modest incentive to GPs generates good response rates for postal questionnaires.

Interventions for the treatment of Morton's neuroma.

BACKGROUND: Morton's neuroma is a common, paroxysmal neuralgia affecting the web spaces of the toes, typically the third. The pain is often so debilitating that patients become anxious about walking or even putting their foot to the ground. Insoles, corticosteroid injections, excision of the nerve, transposition of the nerve and neurolysis of the nerve are commonly used treatments. Their effectiveness is poorly understood. OBJECTIVES: To examine the evidence from randomised controlled trials concerning the effectiveness of interventions in adults with Morton's neuroma. SEARCH STRATEGY: We searched the Cochrane Neuromuscular Disease Group trials register (searched January 2003), MEDLINE (January 1966 to January Week 2 2003), EMBASE (January 1980 to February Week 2 2003), and CINAHL (January 1982 to February Week 1 2003). SELECTION CRITERIA: Randomised or quasi-randomised (methods of allocating participants to an intervention which were not strictly random e.g. date of birth, hospital record, number alternation) controlled trials of interventions for Morton's neuroma were selected. Studies where participants were not randomised into intervention groups were excluded. DATA COLLECTION AND ANALYSIS: Two reviewers selected trials for inclusion in the review, assessed their methodological quality and extracted data independently. MAIN RESULTS: Three trials involving 121 people were included. There is, at most, a very limited indication that transposition of the transected plantar digital nerve may yield better results than standard resection of the nerve in the long term. There is no evidence to support the use of supinatory insoles. There are, at best, very limited indications to suggest that dorsal incisions for resection of the plantar digital nerve may result in less symptomatic post-operative scars when compared to plantar excision of the nerve. REVIEWERS' CONCLUSIONS: There is insufficient evidence with which to assess the effectiveness of surgical and non-surgical interventions for Morton's neuroma. Well designed trials are needed to begin to establish an evidence base for the treatment of Morton's neuroma pain.

Evidence for possible interactions between PLP and DM20 within the myelin sheath.

PLP and its smaller DM20 isoform constitute the major proteins of CNS myelin. Previous studies indicated a role for the proteins in maintaining the intraperiod line of the myelin sheath and the integrity of axons and suggested that both isoforms were necessary to provide these functions. The present study shows that each isoform is capable individually of inserting into compact myelin. Employing chromatographic extraction procedures designed to maintain the natural conformation of the proteins we found that most PLP and DM20 remained associated. Using an antibody specific to the PLP isoform, we were able to co-immunoprecipitate DM20 from the major fraction of the extracted equine myelin and from mouse native whole myelin. We suggest that PLP and DM20 may form a hetero-oligomeric complex within the myelin sheath, probably in association with specific lipids and that this arrangement is essential for the normal structure of myelin and axons.

Influence of ruminal water-loading on renal sodium excretion and water intake following feeding in sheep.

We investigated the effect of ruminal water loading before feeding on the natriuretic and drinking responses that follow feeding. Six sheep fed 800 g of chaff drank 1360 +/- 150 mL during the 5 h immediately following feeding and increased renal Na excretion. Plasma Na concentration increased by 4 mmol L (-1) and plasma osmolality by 9 mosmol kg (-1) within 1.5 h and remained elevated. A rumen load of water administered before feeding prevented the increases in plasma Na and osmolality without affecting feeding. The natriuresis, water drinking and vasopressin secretion in response to feeding were abolished. Total sodium excreted during the experiment was halved in water-loaded animals compared with untreated animals (30.4 +/- 2.1 mmol (-1) cf. 63.8 +/- 2.9 mmol-1; P < 0.01). Ruminal loading with isotonic saline caused a 33% reduction in postprandial drinking, however, reducing cerebrospinal fluid NaCl concentration abolished postprandial drinking and natriuresis. Intravenous infusion of isotonic dextran appeared to delay the onset of water intake without changing the total volume of water drunk, suggesting a role of plasma volume in initiating drinking. We conclude from the data that central osmoregulatory mechanisms that include increased sodium excretion as well as thirst and vasopressin release are activated following food intake by sheep.

Imprinting as a rapid technique for assessing the morphology of the central nervous system by immunofluorescence.

This paper describes a technique that has been developed to assess the in vivo morphology of central nervous system (CNS) tissue by immunofluorescence. This technique permits the study of tissue that is mainly just a monolayer of cells. Unlike routine cryosections that are much thicker (10-15 microm), imprinting does not section the cells, but can result in the detachment of whole cells onto a glass surface for subsequent staining. The imprinting technique is simple and rapid and does not require prior fixation or embedding of the tissue. It has been used to evaluate antigens expressed at the cell surface, in myelin and in the cytoskeleton in the studies of normal and myelin mutant mice. Using the imprinting/immunofluorescence technique one can now assay the genotype of mouse strains that differ in their expression of cell surface antigens within 2 h.

A proteolipid protein-specific pre-mRNA (Ppm-1) contains intron 3 and is up-regulated during myelination in the CNS.

Alternative splicing of the precursor for messenger RNA (pre-mRNA) is a common process utilised by higher eukaryotes to modulate gene expression. A single primary transcript may generate several proteins with distinct functions, expressed in tissue-specific, developmental patterns. This article describes an oligodendrocyte-specific pre-mRNA product of proteolipid protein gene (P/p) transcription, which is the precursor for P/p but not Dm20 mRNA in the CNS. This P/p-specific pre-mRNA (Ppm-1) includes the intact intron 3 of the P/p gene. It is first expressed during active myelination, and it localises to the nucleus of oligodendrocytes, in both normal and jimpy (jp) murine CNS. In addition to mouse, Ppm-1 is found also in rat and dog, but not toad or trout. Our work suggests that alternative splicing of the P/p gene primary transcript follows a branching pattern, resulting in the presence of at least one P/p isoform-specific pre-mRNA molecule, Ppm-1. Therefore, Dm20 mRNA may be the product of a divergent set of pre-mRNA splicing events.

Distinct phenotypes associated with increasing dosage of the PLP gene: implications for CMT1A due to PMP22 gene duplication.

Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.

The early phenotype associated with the jimpy mutation of the proteolipid protein gene.

The jimpy mutation of the X-linked proteolipid protein (Plp) gene causes dysmyelination and premature death of the mice. The established phenotype is characterised by severe hypomyelination, increased numbers of dead oligodendrocytes and astrocytosis. The purpose of this study was to define the earliest cellular abnormalities in the cervical spinal cord. We find that on the first and third postnatal days the amount of myelin in jimpy spinal cord is approximately 20% of wild-type. However, the total glial cell density, the number of dead glial cells and the number and distribution of Plp-positive cells, as assessed by in situ hybridization, are similar to wild-type during the first week of life. Immunostaining of cryosections has identified that jimpy spinal cords express on schedule, a variety of antigens associated with mature oligodendrocytes. Dissociated oligodendrocytes, cultured for 18 hours to reflect their in vivo differentiation, express MBP and surface myelin-associated glycoprotein at the same frequency as wild-type. By comparison, the proportion of jimpy oligodendrocytes expressing surface myelin/oligodendrocyte glycoprotein is reduced by approximately 34%. In vivo, however, only a small minority of axons is surrounded by a collar of myelin-associated glycoprotein, suggesting that the majority of jimpy oligodendrocytes fail to make appropriate ensheathment of axons. Although the DM20 isoform is expressed in the embryonic CNS prior to myelin formation, the cellular abnormalities appear to correspond to the time at which the Plp isoform becomes predominant. The results suggest that the primary abnormality in jimpy is the inability of oligodendrocytes to properly associate with, and then ensheath, axons and that oligodendrocyte death compounds, rather than initiates, the established phenotype.

Distinct Phenotypes Associated with Increasing Dosage of the PLP Gene: Implications for CMT1A Due to PMP22 Gene Duplication.

Increased dosage of the proteolipid protein (Plp) gene causes CNS disease (Pelizaeus-Merzbacher disease [PMD]), which has many similarities to disorders of the PNS associated with duplication of the peripheral myelin protein-22 (PMP22) gene locus. Transgenic mice carrying extra copies of the wild-type Plp gene provide a valid model of PMD. Variations in gene dosage can cause a wide range of phenotypes from severe, lethal dysmyelination through late-onset demyelination. A predilection for different fiber diameters may occur within the various phenotypes with dysmyelination being more obvious in large fibers and late-onset degeneration predominantly affecting small fibers. Although the frequency of apoptotic oligodendrocytes is increased with high gene dosage, the number of mature oligodendrocytes appears adequate. Oligodendrocytes in the dysmyelinated CNS express a range of genes typical of mature cells, yet are unable to assemble sufficient myelin. Oligodendrocytes contain abnormal vacuoles and stain intensely for PLP and other proteins such as MAG. The findings suggest that with high gene dosage much of the PLP, and possibly other proteins, is missorted and degraded in the lysosomal system.

Cytoskeletal and nuclear localization of myelin oligodendrocytic basic protein isoforms.

The recently described single copy myelin-associated oligodendrocytic basic protein (Mobp) gene is expressed exclusively in the central nervous system (CNS). The gene encodes a family of small highly basic polypeptides with predicted amino acid lengths of 69, 71, 81, 99 and 170, all of which share a 68 residue amino terminal. Here we report on the subcellular distribution of two of these polypeptides termed MOBP81 and MOBP170 in transiently transfected Cos7 cells using an antibody raised against a region common to all isoforms of MOBP. Additionally, we describe MOBP trafficking in cultured mouse spinal cord oligodendrocytes. Immunostaining for MOBP81 is intense in the perinuclear region and extends throughout the cytoplasm colocalizing with the microtubular cytoskeletal network. Consistent with this we demonstrate that MOBP partitions with the cytoskeletal fraction prepared from myelin. In contrast, although MOBP170 is present in the cytoplasm it does not colocalize with the cytoskeleton and displays a greater variation in distribution. In the majority of transfectants immunostaining is present throughout the karyoplasm but with increased intensity around the nucleolus. Within mouse primary oligodendrocytes endogenous MOBP is present in the cell body and processes colocalizing with the microtubular network. Immunoreactivity is not detectable in the nucleus in these mature oligodendrocytes. These significant differences in MOBP81 and MOBP170 protein kinesis coupled to different expression profiles of their respective message populations may be indicative of both myelin structural and cellular/regulatory functions, respectively, for these polypeptides.

Molecular cloning and transfection studies of M6b-2, a novel splice variant of a member of the PLP-DM20/M6 gene family.

The present study documents the nucleic acid and deduced amino acid sequence of M6b-2, a novel splice variant of the M6b gene, which belongs to the PLP-DM20/M6 gene family. M6b-2 differs from the previously published M6b by a novel 40-amino acid insertion which is characterised by a high proline content, two casein kinase, and one tyrosine kinase consensus sequences. M6b-2 mRNA is enriched in perinatal central nervous system (CNS), and although it declines during development, it does persist into adulthood. Transient transfection studies coupled to secondary structure and hydrophobicity analysis suggest that the novel polypeptide in M6b-2 lies at the cytoplasmic face of the plasma membrane. It is therefore possible that the function(s) of M6b-2 may be regulated intracellularly by phosphorylation during CNS development.