Spreading depression: evidence of five electroencephalogram phases.

Spreading depression (SD), a self-propagating wave of astroglial and neuronal depolarization, is an accompaniment of several neurological disorders including epilepsy. Its well-described features are initial depolarization, followed by EEG flattening. In this in vivo study in awake animals, the relationship of SDs to epileptiform activity was re-examined. We assessed SDs generated by mechanical stimulation and by metabolic inhibition with fluorocitrate. In addition to identifying prolonged EEG depression, we identified two periods, one prior to and another during depression, characterized by increases in power of specific frequencies that were sometimes associated with epileptiform discharges. The first period was characterized by ripple activity close to the induction site (88% of SDs with intracortical electrodes). The second period was characterized by localized low-frequency spikes (100% with dural screw electrodes, 65% with intracortical electrodes). By using fluorocitrate to induce SDs, the initial period was also characterized by runs of spikes (52%). Finally, with SDs induced by both methods, there was a period at the end of depression when additional, unprovoked SDs occurred (20%). Five stages of SD were defined by these phenomena, in the order: excitation, depression, excitation, depression, SD, with metabolic inhibition enhancing the expression of epileptiform spiking.

Thinking activates EMG in scalp electrical recordings.

OBJECTIVE: Fast electrical rhythms in the gamma range (30-100Hz) in scalp (but not intracranial) recordings are predominantly due to electromyographic (EMG) activity. We hypothesized that increased EMG activity would be augmented by mental tasks in proportion to task difficulty and the requirement of these tasks for motor or visuo-motor output. METHODS: EEG was recorded in 98 subjects whilst performing cognitive tasks and analysed to generate power spectra. In four other subjects, neuromuscular blockade was achieved pharmacologically providing EMG-free spectra of EEG at rest and during mental tasks. RESULTS: In comparison to the paralysed condition, power of scalp electrical recordings in the gamma range varied in distribution, being maximal adjacent to cranial or cervical musculature. There were non-significant changes in mean gamma range activity due to mental tasks in paralysed subjects. In normal subjects, increases in scalp electrical activity were observed during tasks, without relationship to task difficulty, but with tasks involving limb- or eye-movement having higher power. CONCLUSIONS: Electrical rhythms in the gamma frequency range recorded from the scalp are inducible by mental activity and are largely due to EMG un-related to cognitive effort. EMG varies with requirements for somatic or ocular movement more than task difficulty. SIGNIFICANCE: Severe restrictions exist on utilizing scalp recordings for high frequency EEG.

Cell swelling precedes seizures induced by inhibition of astrocytic metabolism.

It is currently unknown what processes take place at the interface between non-ictal and ictal activity during seizure initiation. In this study, using paralysed awake rats, we focally inhibited astrocytic metabolism with fluorocitrate (FC), causing seizures. We measured changes in electroencephalogram (EEG) (0-300 Hz), and extracellular ion-concentrations during ictal onsets defining possible relationships with impedance-determined cell swelling. In animals showing ictal activity (69%) there were spike-wave discharges, spike-wave discharges followed by spreading depression and spreading depression without any discharges. In a high proportion of spike-wave discharges (>95%), just prior to the first spike-wave discharge, there was a decrease in the volume of the extracellular space. Following the initiation of cell swelling and prior to discharges, there were increases in high-frequency (150-300 Hz) EEG activity, increases in extracellular potassium- and decreases in extracellular calcium-concentrations. We suggest that EEG and ionic changes are not causative of cell swelling. Cell swelling due to metabolic failure in astrocytes at the injected site may release excitatory amino acids. At the same time, our results suggest ion homeostasis is not maintained and increased neuronal excitability and synchronisation occur. These could be the drivers changing normal brain activity into ictal activity.

Scalp electrical recording during paralysis: quantitative evidence that EEG frequencies above 20 Hz are contaminated by EMG.

OBJECTIVE: To identify the possible contribution of electromyogram (EMG) to scalp electroencephalogram (EEG) rhythms at rest and induced or evoked by cognitive tasks. METHODS: Scalp EEG recordings were made on two subjects in presence and absence of complete neuromuscular blockade, sparing the dominant arm. The subjects undertook cognitive tasks in both states to allow direct comparison of electrical recordings. RESULTS: EEG rhythms in the paralysed state differed significantly compared with the unparalysed state, with 10- to 200-fold differences in the power of frequencies above 20 Hz during paralysis. CONCLUSIONS: Most of the scalp EEG recording above 20 Hz is of EMG origin. Previous studies measuring gamma EEG need to be re-evaluated. SIGNIFICANCE: This has a significant impact on measurements of gamma rhythms from the scalp EEG in unparalysed humans. It is to be hoped that signal separation methods will be able to rectify this situation.

Standardisation of sheep model for endodontic regeneration/revitalisation research.

OBJECTIVE: Different endodontic regeneration/revitalisation protocols have been suggested for the treatment of immature permanent teeth with pulp necrosis. Many aspects of these protocols require further investigating necessitating a suitable standardised animal model for research purposes. The focus of this study was to examine the anatomy and histology of sheep teeth at different stages of development to find an appropriate dental age for endodontic regeneration/revitalisation research. DESIGN: Sheep teeth at mature and immature dental ages were investigated. Standardized radiography, computed tomography, and histology were used to measure root length, apical-third dentine thickness and apex diameter, and to evaluate tissue development stages. RESULTS: A mature sheep tooth has an apical area which consists of a major foramen, intermediate dilatation and minor foramen. From the time of eruption to maturation no major changes occur in the incisor root lengths, but the apical foramen width decreases and the dentinal wall thickness increases. The two-tooth age exhibited the most similar features to that of an immature permanent human tooth. CONCLUSION: Sheep appears to be an appropriate animal model for endodontic regeneration/revitalization research with similar dimension and characteristics to human anterior teeth. Each dental age has its advantages and disadvantages. The two-tooth age showed the most favourable criteria making this age the most suitable for in vivo regeneration/revitalisation research.

Exposure of fibrinogen-adherent platelets to plasma proteins: a new method for studying protein interactions with platelets.

To understand the effects of mediators in coagulating blood at biomaterial surfaces, it is important to use methods that resemble the normal sequence of events in wound healing around implants. The initial adhesion of platelets from whole blood onto material surfaces is mediated by the fibrinogen receptor glycoprotein IIb/IIIa, as shown in a previous study (Broberg et al., J Lab Clin Med 2002; 139:163-172). In this study, isolated platelets were adhered to fibrinogen and exposed to IgG, von Willebrand factor, or thrombin. The response was detected as the number of adherent platelets, the spreading of platelets, the exposure of CD62P (P-selectin), and the release of platelet factor 4 (PF4), ADP, and ATP. These results were compared to the response of platelets adhering to surfaces coated with the same proteins. Fibrinogen-adherent platelets exposed to thrombin generated the significantly highest exposure of CD62P and release of PF4, ADP, and ATP. When platelets were adhered to different protein coatings, von Willebrand factor generated the most CD62P exposure, IgG generated the most PF4 release, and thrombin generated the highest concentration of ADP. These results indicate that protein interactions with platelets may generate different results, depending on the mode of protein exposure.

Novel in vivo method for evaluation of healing around implants in bone.

A material implanted in bone is always inserted into coagulating blood. Protein and cell interactions during this initial implantation time will govern later healing. Many studies have focused on the tissue surrounding implants. We have developed a method for evaluation of healing around implants in bone by studying cells adhering to the implant surface. Hydrophilic titanium discs were inserted into rat tibiae. Samples were retrieved after 1, 2, 4, and 8 days of implantation and were analyzed by fluorescence microscopy techniques and scanning electron microscopy. Both proliferating and apoptotic cells were found on the surface. Generally, cells closest to the implant surface were nonviable whereas cells in the fibrin network a distance from the surface were viable. Bone morphogenetic protein-2 (BMP-2) is an osteogenic substance. An increase in BMP-2-positive cells was seen during the implantation period, and a population of large BMP-2-positive cells appeared on the surface after 4 days of implantation. The method developed here is a suitable tool for rapid evaluation of the initial healing around implant material.

Preseizure increased gamma electroencephalographic activity has no effect on extracellular potassium or calcium.

Extracellular ion concentrations change during seizures in seizure models. [K(+)](o) increases and [Ca(2+)](o) decreases, resulting from population discharges, enhanced neuronal excitability, though not obviously before seizure onset. In acute pharmacological epilepsy models, there are striking increases in preictal high-frequency (gamma) electroencephalographic (EEG) activity. It is not known whether enhanced gamma EEG results in ionic changes, because gamma and ions have not been measured simultaneously. In this study, unanesthetized, paralyzed rats were given intravenous injections of kainic acid or picrotoxin to induce EEG discharges. Changes in EEG, [K(+)](o), and [Ca(2+)](o) in cortex and hippocampus were recorded. Kainic acid caused small [K(+)](o) fluctuations, without a temporal relationship of these with increased gamma EEG or with onset of discharges. Gamma EEG increases after picrotoxin also failed to affect [K(+)](o) and [Ca(2+)](o). Picrotoxin-induced electrical discharges led to [K(+)](o) rises of >9 mM and [Ca(2+)](o) falls of 0.1-0.2 mM. Kainic acid-induced discharges generated only moderate (2-3 mM) rises in [K(+)](o) and no changes in [Ca(2+)](o). In both models, there were large potassium rises (15-80 mM) and calcium falls (>0.5 mM), suggesting spreading depressions. Small [K(+)](o) fluctuations after kainic acid are consistent with disruption in potassium homeostasis, possibly because of depolarization of astrocytes. To reveal possible latent [K(+)](o) or [Ca(2+)](o) changes, we injected fluorocitrate intracortically to impair astrocytic function, before administering picrotoxin. Even fluorocitrate did not cause gamma-related ion changes but did cause low-magnitude, transient, potassium increases and slower potassium homeostasis during discharges, minor changes consistent with involvement of both astrocytes and neurons in [K(+)](o) regulation. (c) 2007 Wiley-Liss, Inc.

GpIIb/IIIa is the main receptor for initial platelet adhesion to glass and titanium surfaces in contact with whole blood.

Platelets are the first cells to adhere to a surface in contact with blood and are capable of mediating several different responses after contact with different protein-coated surfaces. They are the main source of growth factors such as platelet-derived growth factor and are therefore important in the healing process. In this study, initial platelet adhesion to and spread on hydrophilic and hydrophobic (methylized) glass and titanium with similar wettability were investigated. Whole coagulating blood was used to simulate the in vivo situation shortly after implantation, in which bleeding precedes inflammation and wound healing. Several different antibodies directed against platelet integrins and receptors (CD9, FcgammaRII, GPIIb/IIIa, vitronectin receptor, GPIb/V/IX) were used in an attempt to block platelet adhesion to the surfaces. Immunofluorescence results show that initial platelet adhesion to all the surfaces we investigated can be almost completely inhibited (approximately 95%) by clone M148, an antibody against the GPIIb/IIIa complex (integrin alpha(IIb)beta(3); CD41/CD61), but not with other antibodies to the separate parts of the integrin. Antibodies known to inhibit fibrinogen binding to GPIIb/IIIa after adenosine diphosphate- and collagen- induced aggregation had very little effect on initial platelet adhesion. None of the other integrins were found to have such an effect on initial platelet adhesion. Antibody clone M148 was furthermore found to inhibit platelet spreading. This study shows that regardless of wettability and the biomaterial used, initial adhesion of platelets appears to be mediated by GPIIb/IIIa binding to surface adsorbed fibrinogen.

Fluorocitrate-mediated astroglial dysfunction causes seizures.

A role for astroglia in epileptogenesis has been hypothesised but is not established. Low doses of fluorocitrate specifically and reversibly disrupt astroglial metabolism by blocking aconitase, an enzyme integral to the tricarboxylic acid cycle. We used cerebral cortex injections of fluorocitrate, at a dose that we demonstrated to inhibit astroglial metabolism selectively, to determine whether astroglial disturbances lead to seizures. Rats were halothane-anesthetized, and 0.8 nmol of sodium fluorocitrate was injected into the cerebral cortex. Extradural electroencephalogram (EEG) electrodes were implanted, after which the anesthesia was ceased and the animals were observed. In all experiments, 14 of 15 fluorocitrate-treated animals exhibited epileptiform EEG discharges, with some animals exhibiting convulsive seizures. Discharges commenced as early as 30 min postfluorocitrate injection. Intraperitoneal octanol, but not halothane by inhalation, given to test the possible participation of gap junctions in EEG discharge generation, blocked or delayed the occurrence of discharges after fluorocitrate. These results indicate that focal cerebrocortical astroglial dysfunction leads to focal epileptiform discharges and sometimes to convulsive seizures and that the process possibly depends on effects mediated by gap junctions.