Are patient educational resources effective at deterring stroke survivors from considering experimental stem cell treatments? A randomized controlled trial.

OBJECTIVE: To evaluate whether online resources developed to educate people about the risks associated with experimental stem cell (SC) treatments influence stroke survivors' attitudes about the safety and effectiveness of these treatments. METHODS: Adult stroke survivors who had not previously received SC treatments (N = 112) were recruited from international stroke advocacy/support groups for a prospective, parallel-group randomized controlled trial. Participants indicated whether they were considering SC treatments (yes/no) prior to, immediately following, and 30-days after reading/viewing the International Society for Stem Cell Research booklet or Stem Cell Network video. Participant attitudes regarding the safety, effectiveness, accessibility and affordability of SC treatments were examined on each occasion, and compared to those of a waitlist control group. RESULTS: Significantly fewer participants were considering SC treatments immediately after reading the SC research booklet (p =.031), although neither intervention had any impact after 30-days (p >.05). Waitlist and intervention groups reported positive attitudes toward SC treatments at each assessment. CONCLUSIONS: Stroke survivor attitudes toward SC treatments were initially influenced by the patient booklet, however these changes were not maintained. PRACTICAL IMPLICATIONS: Clinicians are encouraged to initiate discussions about experimental SC treatments during inpatient rehabilitation and to reinforce the risks throughout subsequent care.

Alterations in anxiety and social behaviour in Npas4 deficient mice following photochemically-induced focal cortical stroke.

In addition to causing widespread cell death and loss of brain function, cerebral ischaemia also induces extensive neuroplasticity. In humans, stroke is often accompanied by severe cognitive and psychiatric changes that are thought to arise as a consequence of this infarct-induced remodelling. A candidate for producing these post-stroke neuropsychiatric changes is Npas4, an activity-dependent transcription factor involved in synaptic plasticity whose expression is aberrantly up-regulated following ischaemic injury. In this study we investigated the role of Npas4 in modulating these stroke-induced neuropsychiatric responses by comparing the performance of wildtype and Npas4-/- mice in various cognitive and behavioural tasks in a photochemical model of focal cortical stroke. We show that this stroke model results in impaired spatial recognition memory and a reduction in despair-like behaviour that affect both genotypes to a similar degree. Moreover, mice lacking Npas4 also show differences in some aspects of post-stroke sociability and anxiety. Specifically, we show that while stroke had no effect on anxiety levels in wildtype mice, Npas4-/- mice became significantly more anxious following stroke. In addition, Npas4-/- mice retained a greater level of sociability in the acute post-stroke period in comparison to their wildtype littermates. Thus, our findings suggest that Npas4 may be involved in post-stroke psychiatric changes related to anxiety and sociability.

Inflammasome gene expression alterations in Staphylococcus aureus biofilm-associated chronic rhinosinusitis.

BACKGROUND: The role of inflammasomes in chronic inflammation has been the subject of intense research in recent years. Chronic rhinosinusitis (CRS), a persistent inflammatory disease, continues to be investigated hoping that a clearer pathophysiologic description will guide discovery of future treatment modalities. This study investigates the role of inflammasome complexes in CRS patients with Staphylococcus aureus biofilm infection, a key culprit associated with disease severity and recalcitrance. METHODOLOGY: Sinonasal tissue samples were collected from CRS patients with (P+) and without (P-) polyps and controls. S. aureus biofilm status was obtained using fluorescence in situ hybridization and classified as biofilm positive (B+) or negative (B-). RNA was analysed using a Human Inflammasome PCR array, profiling the expression of 84 genes involved in inflammasome function. RESULTS: Sixteen samples were obtained: 5 B+P+, 5 B-P- and 6 controls. Comparing B+P+ vs. controls showed the greatest number of differentially expressed genes. In particular, Absent in Melanoma 2 (AIM2) was consistently and significantly up-regulated in the B+P+ vs. B-P- and controls. In contrast, when comparing the B-P- vs. controls, no genes showed significant changes. CONCLUSION: Our results indicate the involvement of inflammasome complexes and their signalling pathways in CRS patients with polyps and S. aureus biofilms. In particular, AIM2, activated by intracellular double-stranded DNA, is up-regulated in this group, implying that S. aureus may play a role in intracellular triggering of the inflammasome response. Studies with further patient stratification and assessing corresponding protein expression are needed to further characterize the role of inflammasomes in CRS.

A patient with anaphylaxis after alteplase infusion.

Anaphylaxis to alteplase is a rare but reported complication of intravenous thrombolysis. We report a patient with a documented episode of anaphylaxis that occurred following an initial bolus and a subsequent delayed infusion of alteplase for thrombolysis of acute ischaemic stroke. The patient was treated with hydrocortisone, adrenaline, prochlorperazine and ranitidine, as per the hospital anaphylaxis protocol, intubation and admission to the intensive care unit. Serum tryptase levels performed during the anaphylactic event (at the end of the infusion) and 1.5 hours later showed an increase of 2 µg/L, suggestive of an anaphylactic reaction. Anaphylaxis remains largely a clinical diagnosis even in the absence of an elevated serum tryptase. The patient would benefit from further allergen testing given the severity of the reaction to alteplase. We report this patient to indicate that although rare, anaphylaxis is a recognised adverse event following alteplase. In the case of any symptoms suggestive of a minor anaphylactic reaction to alteplase, further infusion should be ceased to avoid a dose dependent major reaction.

An essential function for the centrosomal protein NEDD1 in zebrafish development.

The centrosome is the primary microtubule organising centre of the cell. It is composed of many proteins, some of which make up the core of the centrosome, whereas others are used for specific functions. Although the cellular roles of many centrosomal proteins are well defined, much less is known about their functions and the role of the centrosome in development. In this study we investigated the function of NEDD1, a critical component of the centrosome essential for microtubule nucleation, in zebrafish (Danio rerio) development. The zebrafish homologue of NEDD1 (zNEDD1) was cloned and found to have a similar localisation and function to mammalian NEDD1. We show that zNEDD1 is essential for survival, as a high level of knockdown was embryonic lethal. Partial knockdown of zNEDD1 caused abnormalities including an increase in mitotic and apoptotic cells. Pronounced phenotypic defects were seen in the brain, with a lack of defined brain structures, incomplete neural tube formation and a disorganisation of neurons. In addition, we show that a reduction in zNEDD1 resulted in the loss of gamma-tubulin at the centrosome. Our data thus demonstrate that zNEDD1 is critical for the recruitment of gamma-tubulin to the centrosome, and is essential for the proper development of zebrafish.

High EphA3 expressing ophthalmic trigeminal sensory axons are sensitive to ephrin-A5-Fc: implications for lobe specific axon guidance.

The ophthalmic, maxillary and mandibular axon branches of the trigeminal ganglion provide cutaneous sensory innervation to the vertebrate face. In the chick embryo, the trigeminal ganglion is bilobed, with ophthalmic axons projecting from the ophthalmic lobe, while maxillary and mandibular projections emerge from the maxillomandibular lobe. To date, target tissue specific guidance cues that discriminately guide the axon projections from the two trigeminal ganglion lobes are unknown. EphA receptor tyrosine kinases and ephrin-A ligands are excellent candidates for this process as they are known to mediate axon guidance in the developing nervous system. Accordingly, the expression of EphAs and ephrin-As was investigated at stages 13, 15, 20 of chick embryogenesis when peripheral axons from the trigeminal ganglion are pathfinding. EphA3 is expressed highly in the ophthalmic trigeminal ganglion lobe neurons in comparison to maxillomandibular trigeminal ganglion lobe neurons. Furthermore, from stages 13-20 ephrin-A2 and ephrin-A5 ligands are only localized to the mesenchyme of the first branchial arch (maxillary and mandibular processes), the target fields for maxillomandibular trigeminal ganglion axons. We found that ophthalmic and not maxillomandibular lobe axons were responsive to ephrin-A5-Fc utilizing a substratum choice assay. The implication of these results is that EphA3 forward signaling in ophthalmic sensory axons may be an important mechanism in vivo for lobe specific guidance of trigeminal ganglion ophthalmic projections.

EphA4 constitutes a population-specific guidance cue for motor neurons.

Motor neurons in the ventral neural tube project axons specifically to their target muscles in the periphery. Although many of the transcription factors that specify motor neuron cell fates have been characterized, less is understood about the mechanisms that guide motor axons to their correct targets. We show that ectopic expression of EphA4 receptor tyrosine kinase alters the trajectories of a specific population of motor axons in the avian hindlimb. Most motor neurons in the medial portion of the lateral motor column (LMC) extend their axons aberrantly in the dorsal nerve trunk at the level of the crural plexus, in the presence of ectopic EphA4. This misrouting of motor axons is not accompanied by alterations in motor neuron identity, settling patterns in the neural tube, or the fasciculation of spinal nerves. However, ectopic EphA4 axons do make errors in pathway selection during sorting in the plexus at the base of the hindlimb. These results suggest that EphA4 in motor neurons acts as a population-specific guidance cue to control the dorsal trajectory of their axons in the hindlimb.

Expression of EphA4, ephrin-A2 and ephrin-A5 during axon outgrowth to the hindlimb indicates potential roles in pathfinding.

During neural development, spinal motor axons extend in a precise manner from the ventral portion of the developing spinal cord to innervate muscle targets in the limb. Although classical studies in avians have characterized the cellular interactions that influence motor axon pathfinding to the limb, less is known about the molecular mechanisms that mediate this developmental event. Here, we examine the spatiotemporal distributions of the EphA4 receptor tyrosine kinase (RTK) and its cognate ligands, ephrin-A2 and ephrin-A5, on motor neurons, their axons and their pathways to the avian hindlimb to determine whether these molecules may influence axonal projections. The expression patterns of EphA4, ephrin-A2 and ephrin-A5 mRNAs and proteins are highly complex and appear to exhibit some overlap during motor axon outgrowth and pathfinding to the hindlimb, reminiscent of the co-expression of Eph RTKs and ephrins in the retinotectal system. EphA4, similar to the carbohydrate moiety polysialic acid, strikingly marks the main dorsal, but not ventral, nerve trunk after axon sorting at the limb plexus region. Our results suggest that EphA4 RTK and its ligands may influence axon fasciculation and the sorting of axons at the limb plexus, contributing to the correct dorsoventral organization of nerve branches in the hindlimb.

Spinal motor axons and neural crest cells use different molecular guides for segmental migration through the rostral half-somite.

The peripheral nervous system in vertebrates is composed of repeating metameric units of spinal nerves. During development, factors differentially expressed in a rostrocaudal pattern in the somites confine the movement of spinal motor axons and neural crest cells to the rostral half of the somitic sclerotome. The expression patterns of transmembrane ephrin-B ligands and interacting EphB receptors suggest that these proteins are likely candidates for coordinating the segmentation of spinal motor axons and neural crest cells. In vitro, ephrin-B1 has indeed been shown to repel axons extending from the rodent neural tube (Wang & Anderson, 1997). In avians, blocking interactions between EphB3 expressed by neural crest cells and ephrin-B1 localized to the caudal half of the somite in vivo resulted in loss of the rostrocaudal patterning of trunk neural crest migration (Krull et al., 1997). The role of ephrin-B1 in patterning spinal motor axon outgrowth in avian embryos was investigated. Ephrin-B1 protein was found to be expressed in the caudal half-sclerotome and in the dermomyotome at the appropriate time to interact with the EphB2 receptor expressed on spinal motor axons. Treatment of avian embryo explants with soluble ephrin-B1, however, did not perturb the segmental outgrowth of spinal motor axons through the rostral half-somite. In contrast, under the same treatment conditions with soluble ephrin-B1, neural crest cells migrated aberrantly through both rostral and caudal somite halves. These results indicate that the interaction between ephrin-B1 and EphB2 is not required for patterning spinal motor axon segmentation. Even though spinal motor axons traverse the same somitic pathway as neural crest cells, different molecular guidance mechanisms appear to influence their movement.

Motor axon pathfinding in the peripheral nervous system.

Functional motor performance is dependent upon the correct assemblage of neural circuitry, a process initiated during embryonic development. How is the complicated neural circuitry that underlies functional behavior formed? During early stages of development, motor neurons extend their axons in a precise manner to their target destinations where they form fine synaptic connections. This process is not random but rather, highly stereotyped and specific. Results of recent studies indicate that positive and negative molecules influence particular steps in the navigation of motor axons to their targets. These molecules include, but are not limited to, members of the Semaphorin family and their receptors, Neuropilins and Plexins, Slits and their Robo receptors, members of the Eph family, extracellular matrix molecules, Hepatocyte Growth Factor/Scatter Factor, peanut agglutinin-binding glycoproteins, and neural cell adhesion molecule. The developing avian peripheral nervous system has served as an excellent model system for many years for studies of the basic cellular interactions that underlie motor axon pathfinding. The principal advantage for the experimental use of the avian embryo is the ease of access to early developmental events. Fine microsurgical manipulations, difficult at best in mouse embryonic development, are readily accomplished in avian embryos and have provided a powerful approach to unraveling the cellular interactions that govern motor axon pathfinding. These approaches, combined in recent years with molecular biology, have begun to produce critical insights into the mechanisms that sculpt cellular architecture during neural development.

Pax-3 regulates neurogenesis in neural crest-derived precursor cells.

The peripheral nervous system consists of multiple neural lineages derived from the neural crest (NC). Pax-3 is expressed in the NC and when mutated in the splotch mouse (Sp) results in the loss of derivatives from this precursor cell population. We have investigated the role of Pax-3 in regulating the generation of neurons from NC-derived precursor cells in vitro. Pax-3 mRNA in NC cultures is initially expressed in all NC but is subsequently only retained in neurons, suggesting a role in their generation. To determine whether Pax-3 is involved in neuron development, we first examined the generation of sensory-like neurons in NC cultures from Sp mice. Fivefold less sensory-like neurons were generated in NC cultures from Sp homozygous mice as compared to wild-type littermates. The role of Pax-3 in sensory neuron generation was then directly examined in dorsal root ganglia cultures by down-regulating the expression of Pax-3 protein with antisense oligonucleotides. It was found that antisense oligonucleotides inhibited 80-90% of newly generated sensory neurons; however, there was no significant effect on the survival of sensory neurons or the precursor population. These results suggest that Pax-3 has a role in regulating the differentiation of peripheral neurons.

Neural precursor differentiation into astrocytes requires signaling through the leukemia inhibitory factor receptor.

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR-/-) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured in vitro. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the in vivo findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR-/- mice. In addition, monolayers of neural cells from LIFR-/- mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR-/- precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.

Targeted disruption of the low-affinity leukemia inhibitory factor receptor gene causes placental, skeletal, neural and metabolic defects and results in perinatal death.

The low-affinity receptor for leukemia inhibitory factor (LIFR) interacts with gp130 to induce an intracellular signal cascade. The LIFR-gp130 heterodimer is implicated in the function of diverse systems. Normal placentation is disrupted in LIFR mutant animals, which leads to poor intrauterine nutrition but allows fetuses to continue to term. Fetal bone volume is reduced greater than three-fold and the number of osteoclasts is increased six-fold, resulting in severe osteopenia of perinatal bone. Astrocyte numbers are reduced in the spinal cord and brain stem. Late gestation fetal livers contain relatively high stores of glycogen, indicating a metabolic disorder. Hematologic and primordial germ cell compartments appear normal. Pleiotropic defects in the mutant animals preclude survival beyond the day of birth.

Factors regulating the differentiation of neural precursors in the forebrain.

Precursors from the neuroepithelium of the developing cortex and the adult subventricular zone can be cloned in vitro after stimulation with fibroblast growth factor 2 (FGF-2), and they have the potential to give rise to both neurons and glia. The generation of neurons from these clones can be stimulated by either a factor derived from an astrocyte precursor line, Ast-1, or FGF-1. We have shown that neuronal differentiation stimulated by FGF-1 can be inhibited by diacylglycerol lipase inhibitor and mimicked by arachidonic acid, suggesting that the neuronal differentiation is signalled through the phospholipase C gamma pathway. The sequential expression of FGF-2, followed by FGF within the developing forebrain neuroepithelium, fits with the different functions that the two FGFs play in precursor regulation. We have shown that the precursor response to FGF-1 is regulated by a heparan sulphate proteoglycan expressed within the developing neuroepithelium. Precursors restricted to the astrocyte cell lineage can be stimulated by epidermal growth factor or FGF-2F however, the differentiation into glial fibrillary acidic protein-positive astrocytes appears to require a cytokine acting through the leukaemia inhibitory factor-beta receptor.

The engraftment of transplanted primary neuroepithelial cells within the postnatal mouse brain.

Primary neuroepithelial precursor cells carrying the reporter gene lacZ were transplanted into postnatal murine brain and assessed for their engraftment capacity. Freshly dissected precursors, derived from lacZ transgenic embryonic day 10 mouse brain, predominantly engrafted as discrete clusters, whereas the same precursors cultured in vitro with fibroblast growth factor-2, engrafted as single cells within the parenchyma of the hippocampus. Approximately 0.5% of the transplanted cells survived in the host brain for up to 3 months. Many of these cells displayed neuronal and astrocyte morphologies. These observations suggest that transplanted primary precursors derived from the embryonic brain can engraft and commit in situ to a variety of developmental fates.

Video-audio/EEG monitoring in epilepsy--the Queen Elizabeth Hospital experience.

Our experience of using video-audio/EEG monitoring in the diagnosis and management of epilepsy at The Queen Elizabeth Hospital Comprehensive Epilepsy Service from March 1987 to December 1990 is described. We performed 75 long term monitoring studies on a total of 66 patients. Following monitoring, a change in seizure diagnosis was made in 21 of 66 patients (32%). Pseudoseizures were diagnosed in 17 patients. A change in management as a consequence of monitoring occurred in 53 of the 66 patients (80%). The referring neurologists considered that 56 of the 75 studies (75%) were successful. The investigational technique is effective and is particularly useful for the diagnosis of pseudoseizures.