Anti-fibronectin aptamers improve the colonization of chitosan films modified with D-(+) Raffinose by murine osteoblastic cells.

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.

A cytotoxicity study of silicon oxycarbide nanowires as cell scaffold for biomedical applications.

GOAL: Nanowires are promising biomaterials in multiple clinical applications. The goal of this study was to investigate the cytotoxicity of carbon-doped silica nanowires (SiOxCy NWs) on a fibroblastic cell line in vitro. MATERIALS AND METHODS: SiOxCy NWs were grown on Si substrates by CVD process. Murine L929 fibroblasts were cultured in complete DMEM and indirect and direct cytotoxicity tests were performed in agreement with ISO 19003-5, by quantitating cell viability at MTT and chemiluminescent assay. Cell cultures were investigated at Scanning Electron Microscope (SEM) and immunocytochemistry to observe their morphology and investigate cell-NWs interactions. Furthermore, hemocompatibility with Platelet-rich Plasma was assayed at SEM and by ELISA assay. RESULTS: SiOxCy NWs proved biocompatible and did not impair cell proliferation at contact assays. L929 were able to attach on NWs and proliferate. Most interestingly, L929 reorganised the NW scaffold by displacing the nanostructure and creating tunnels within the NW network. NWs moreover did not impair platelet activation and behaved similarly to flat SiO2. CONCLUSIONS: Our data show that SiOxCy NWs did not release cytotoxic species and acted as a viable and adaptable scaffold for fibroblastic cells, thus representing a promising platform for implantable devices.

Improved scaffold biocompatibility through anti-Fibronectin aptamer functionalization.

UNLABELLED: Protein adsorption is the first and decisive step to define cell-biomaterial interaction. Guiding the adsorption of desired protein species may represent a viable approach to promote cell activities conducive to tissue regeneration. The aim of the present study was to investigate whether immobilized anti-Fibronectin aptamers could promote the attachment and growth of osteoblastic cells. Polyethyleneglycole diacrylate/thiolated Hyaluronic Acid hydrogels (PEGDA/tHA) were coated with anti-Fibronectin aptamers. Hydrogel loading and Fibronectin bonding were investigated, through spectrophotometry and Bradford assay. Subsequently, human osteoblasts (hOBs) were cultured on hydrogels for 10days in 2D and 3D cultures. Cells were monitored through microscopy and stained for focal adhesions, microfilaments and nuclei using fluorescence microscopy. Samples were also included in paraffin and stained with Hematoxylin-Eosin. Cell number on hydrogels was quantitated over time. Cell migration into the hydrogels was also studied through Calcein AM staining. Aptamers increased the number of adherent hOBs and their cytoplasm appeared more spread and richer in adhesion complexes than on control hydrogels. Viability assays confirmed that significantly more cells were present on hydrogels in the presence of aptamers, already after 48h of culture. When hOBs were encapsulated into hydrogels, cells were more numerous on aptamer-containing PEGDA-tHA. Cells migrated deeper in the gel in the presence of DNA aptamers, appearing on different focus planes. Our data demonstrate that anti-Fibronectin aptamers promote scaffold enrichment for this protein, thus improving cell adhesion and scaffold colonization. STATEMENT OF SIGNIFICANCE: We believe aptamer coating of biomaterials is a useful and viable approach to improve the performance of scaffold materials for both research and possibly clinical purposes, because different medical devices could be envisaged able to capture bioactive mediators from the patients' blood and concentrate them where they are needed, on the biomaterial itself. At the same time, this technology could be used to confer 3D cell culture scaffold with the ability to store proteins, such as Fibronectin, taking it from the medium and capture what is produced by cells. This is an improvement of traditional biomaterials that can be enriched with exogenous molecules but are not able to selectively capture a desired molecule.

Chitosan scaffold modified with D-(+) raffinose and enriched with thiol-modified gelatin for improved osteoblast adhesion.

The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation. 2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml(-1) BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry. Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured. Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve. Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin. Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation. Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties.

Interactions among pro-inflammatory cytokines, IGF system and thyroid function in pre-pubertal obese subjects.

Obesity is a state of chronic inflammation. Data on IGF system are often discrepant, and their relationships with mediators of inflammation are unknown. Furthermore, changes in thyroid function have been reported. We aimed at investigating the changes in these systems, and verify any relationships among cytokines, IGF system, thyroid function and insulin-insensitivity. Fifty obese pre-pubertal children, and 55 normal-weight subjects comparable for age and sex were enrolled. Serum IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, IL-6 and TNF-alpha were assayed. In obese children insulin, TSH and FT4 were measured also, and the HOMA-IR index was calculated. Increased IGF-II, IL-6 and TNF-alpha, and decreased IGFBP-1 and IGFBP-2 concentrations were found in obese compared to normal-weight children. The IGF-I/IGFBP-3 molar ratio was also reduced in the obese subjects. In the obese children with high HOMA-IR index, IGFBP-1 and -2 serum concentrations were significantly decreased compared with those with normal insulin sensitivity, and in the obese subjects with increased TSH, IGFBP-2 concentrations were lower, and IGFBP-3 levels were higher compared to their counterparts with normal TSH levels. Among the significant correlations, BMISDS was correlated with IGF-II, and TSH. IGF-II concentrations showed a positive relationship with IL-6. TSH was correlated with IGFBP-2 also. The data showed interactions among IL-6, IGF system, insulin sensitivity, and thyroid function with changes being related to the degree of obesity. Chronic inflammation in obese children was confirmed. Some of the changes in the IGF system could be a consequence of insulin resistance and could account also for later complications in obese subjects.

Association of placental insulin, total and activated insulin receptor contents, cortisol and IL-6 concentrations with human birth weight and length: pilot study.

We followed-up, from pregnancy to birth, a group of newborns both IUGR and AGA and we aimed at establishing placental biochemical determinants of birth weight and length. Insulin, total and activated insulin receptor contents (IR), cortisol and IL-6 placental concentrations were assayed in 23 IUGR and 37 AGA subjects at birth, and a multiple regression model was designed and applied to assess the significant biochemical determinants of birth size. IL-6 and activated insulin receptor content were significantly increased in IUGR, whereas insulin, total insulin receptor content, and cortisol placental concentrations were similar in IUGR and AGA. Placental cortisol concentration was found to be significantly and negatively related with both birth length (0.778, P<0.001) and weight (0.508, P<0.008). A negative effect of IL-6 placental concentration was found on birth length (P<0.002). For the first time we provide evidence of a negative association of placental cortisol and IL-6 concentrations on birth size.

Placental cortisol and cord serum IGFBP-2 concentrations are important determinants of postnatal weight gain.

There is a need to identify simple biochemical markers at birth that may predict subjects at risk of growth failure and metabolic complications in later life. Limited research to date has been performed on relationships of specific biochemical determinants at birth with postnatal weight gain and growth. We proposed to establish whether placental cortisol and IL-6 concentrations and cord serum IGF-II and IGFBP-2 concentrations influenced postnatal growth. We followed up from pregnancy 23 IUGR and 37 AGA subjects, and determined placental cortisol and IL-6 concentrations, and cord serum IGF-II, and IGFBP-2 concentrations at birth. We obtained height and weight measurements at 3, 6, 12, 24 months and 5 years of age in 20 IUGR and 15 AGA subjects of comparable gestational age. A multiple linear regression model was designed to establish the effect of the placental and cord serum peptides on postnatal linear growth and weight gain. All IUGR subjects had catch-up growth before 2 years of age. Placental cortisol concentration correlated positively with weight gain during the first 5 years of postnatal growth (P<0.05). Subjects with the highest placental cortisol concentrations were those who showed a greater increase in weight. Cord serum IGFBP-2 concentrations correlated positively with weight gain throughout the 5 year observation period (P:0.003). The subjects with the highest concentrations showed a greater weight gain. Placental cortisol and cord serum IGFBP-2 concentrations were related to postnatal weight gain, suggesting that the fetal environment has long-term effects on growth.

Optoelectronic reservoir computing.

Reservoir computing is a recently introduced, highly efficient bio-inspired approach for processing time dependent data. The basic scheme of reservoir computing consists of a non linear recurrent dynamical system coupled to a single input layer and a single output layer. Within these constraints many implementations are possible. Here we report an optoelectronic implementation of reservoir computing based on a recently proposed architecture consisting of a single non linear node and a delay line. Our implementation is sufficiently fast for real time information processing. We illustrate its performance on tasks of practical importance such as nonlinear channel equalization and speech recognition, and obtain results comparable to state of the art digital implementations.

Impairment of insulin receptor signal transduction in placentas of intra-uterine growth-restricted newborns and its relationship with fetal growth.

OBJECTIVE: Intra-uterine growth restriction (IUGR) is related to a higher incidence of type 2 diabetes mellitus. We previously reported reduced adiponectin and increased interleukin 6 (IL6) concentrations in IUGR placentas, which are features of insulin resistance. We aimed to investigate placental insulin receptor (IR) function and activation in human placenta and subsequently the relationships of insulin signalling peptides with placental protein content in IL6, insulin, resistin and adiponectin, and with parameters of fetal growth. DESIGN AND METHODS: Whole villous tissue was collected from 18 IUGR and 24 appropriate for gestational age (AGA) placentas of comparable gestational age. Insulin signalling peptides, suppressors of cytokine signalling-2 (SOCS2), insulin, adiponectin, resistin, and IL6 concentrations were determined by using western immunoblotting or specific research kits. RESULTS: The amount of total IR was similar in both groups but activated IR significantly higher in IUGR. Total IR substrate-1 (IRS1) was increased in IUGR, whereas total IRS2 and activated IRS1 were similar. AKT content was reduced and activated AKT was undetectable in IUGR placentas. c-Jun N-terminal kinase content was reduced in IUGR. Total and activated ERK1/2 was similar in IUGR and AGA groups, and total SOCS2 was increased in IUGR. IL6 lysate concentrations correlated with AKT content and activated IR. Correlations were found also with adiponectin and resistin. SOCS2 correlated negatively with all growth parameters at birth. CONCLUSIONS: IR was more activated in placentas of IUGR compared with AGA; however, signal transduction downstream of the receptor was impaired. The increase in activated IR could be in favour of a compensatory mechanism to increase insulin sensitivity. Close relationships of insulin action in placenta with fetal growth were shown.

Doping-induced conductivity transitions in molecular layers of polyaniline: detailed structural study.

We report detailed structural investigations, by synchrotron X-ray reflectivity (XRR), grazing incidence diffraction (GID), and space-resolved grazing incidence X-ray-induced fluorescence (GIXF), on the structure of molecular layers of polyaniline (PANI) that can be converted from insulating to conducting state simply by doping. We first address the simpler, but more intriguing, system, i.e., a floating monolayer of PANI on different subphases, for which we found a typical thickness of 28(1) A, not much affected by the doping process. For the doped film we also found an internal lateral structure, with in-plane spacing of 3.5 A-albeit with a small coherence of 3-4 repeat units only-compatible with face-to-face interchain stacking of phenyl rings, in agreement with the literature. By GIXF we could confirm the crucial role of Cl(-) intercalation in the doping process of the PANI film: under doping conditions (0.1 M HCl subphase) the Cl(-) intake is 8 times larger than in nondoping conditions (0.1 M KCl subphase). Multilayers transferred onto solid substrate were studied also as a function of the applied voltage, as this system constitutes the core of an electrochemically controlled device whose strongly nonlinear characteristic make it useful for applications to adaptive networks for complex information processing. By the application of an electrostatic field of 140 V/m, Cl ionic migration was observed confined to the polymeric film surface.

Riedel's thyroiditis: clinical, pathological and imaging features.

Riedel's thyroiditis (RT) is an extremely rare form of chronic thyroiditis, characterised by a fibroinflammatory process which partially destroys the thyroid, often involving surrounding tissues. We describe an unusual case of RT in a 38-year-old woman, and discuss its typical clinical, pathological, ultrasound, radionuclide scanning and magnetic resonance findings. We conclude that the diagnosis of RT is highly suggestive in the presence of a hard gland (or nodule), fixed to adjacent structures; 'cold' on Tc99 scan; hypoechoic with absence of vascular flow, invading the adjacent neck structures on ultrasound; hypointense in T1- and T2-weighted MR images; and showing fibrous tissue fragments with inflammatory cells at cytological examination. Thyroidectomy has to be performed to confirm the diagnosis and to relieve pressure symptoms.

Sleep in Lennox-Gastaut syndrome: the role of the cyclic alternating pattern (CAP) in the gate control of clinical seizures and generalized polyspikes.

Non-rapid eye movement (NREM) sleep contains periods of arousal instability (cyclic alternating pattern or CAP) and periods of arousal stability (non-CAP). During CAP, arousal oscillates between higher (phase A) and lower (phase B) levels of activation. We evaluated the relationship between CAP and the occurrence of epileptic events, i.e. clinical seizures and generalized interictal discharges, during sleep in 10 patients with Lennox-Gastaut syndrome (LGS). The macro- and microstructure of sleep of 10 attended overnight polysomnograms were analyzed. Compared with 10 age- and gender-matched controls, patients with LGS had significantly less stage 2 and REM sleep and higher amounts of CAP rate (68% vs. 33%; P<0.0001). The number of generalized polyspike bursts per hour of sleep was highest in slow wave sleep (226.5+/-57.6) and lowest in REM sleep (3.9+/-1.5). The polyspike burst frequency was significantly greater (P<0.017) during CAP (213.2+/-60.1) than during non-CAP (100.3+/-40), and within CAP, generalized polyspikes occurred more often (P=0.005) during phase A (461.1+/-127.2) than during phase B (6.1+/-1.9). The total amount of generalized polyspike bursts identified in NREM sleep correlated positively both with the number of A phases containing at least one generalized polyspike (P=0.005) and with the mean number of polyspikes within each of these A phases (P<0.0001). Nocturnal clinical seizures occurred in 8 of the 10 patients and showed a similar trend. We conclude from our results that CAP modulates the occurrence of both clinical seizures and generalized epileptic discharges in LGS by means of a gate-control mechanism: an independent spike generator is inhibited in phase B and non-CAP and bursts with its intrinsic activity in phase A.

Combined influence of cyclic arousability and EEG synchrony on generalized interictal discharges within the sleep cycle.

PURPOSE: to analyze the activating role of cyclic alternating pattern (CAP) and EEG synchrony on generalized interictal paroxysms in the first part of the night, when all sleep patterns are represented. METHODS: nocturnal polysomnographic investigation was accomplished on a randomized series of 18 subjects with an active form of primary generalized epilepsy (PGE), but only six patients showed a complete and regular profile of the first two sleep cycles (SCs). Completeness and regularity of the selected SCs consisted in the absence of intervening wakefulness, in the presence of all sleep stages, and in the identification of three main units, (a) a descending branch, dominated by the build-up of EEG synchrony in the transition from light to deep non-rapid eye movement (NREM) sleep; (b) a trough, where the magnitude of EEG synchrony is greatest and gives rise to stages 3 and 4; (c) an ascending branch characterized by a decrease of EEG synchrony preceding the onset of rapid eye movement (REM) sleep. Generalized paroxysms were evaluated in terms of discharge rates (number of interictal bursts per minute of sleep) and distribution within the investigated sleep parameters. RESULTS: the discharge rates decreased from SC1 to SC2, with higher values quantified during NREM sleep (mean, 2.8) compared with REM sleep (mean, 0.8). Both SCs showed a progressive decrease of activation across the three units, from the highest discharge rates reached during the descending branches (mean, 3.6) to the more attenuated discharge rates during the troughs (mean, 2.4) down to the lowest rates during the ascending limbs (mean, 1.1). The magnitude of activation during the descending branches was closely related to the CAP condition (mean, 5.2) and to the powerful effect of phase A (mean, 13.9). The great majority (82%) of EEG discharges occurring in phase A were distributed within the A1 subtypes (identified by sequences of k-complexes or delta bursts). CONCLUSIONS: within the first two SCs, the features of NREM sleep endowed with the major activating power on generalized bursts are represented by the rise of EEG synchrony (descending branch) and by the A phases of CAP involved in the regulation of its build-up.

[Neurophysiological basis of insomnia: role of cyclic alternating patterns]. / Les bases neurophysiologiques de l'insomnie: le rôle des tracés alternant cycliques (CAP).

During non-REM (NREM) sleep it is possible to identify two complementary conditions of arousal stability and arousal instability. Unstable sleep, which can be detected in all stages, is expressed by the recurrence of arousal complexes (sequences of K-complexes, delta bursts, K-alpha, conventional arousals), which translate a brief (10-15 s) activation of the sleeping brain. These repetitive arousal complexes compose the cyclic alternating pattern (CAP). During stable NREM sleep, arousal complexes are rare or absent and the EEG lacks any cyclic pattern (non-CAP). CAP is a spontaneous feature of normal sleep as it is typically involved in stage changes and nocturnal motor activity. The physiological amount of CAP rate (ratio of CAP time to NREM sleep time) varies with age according to a U-shape curve. Within these ranges, sleep is perceived as continuous and restorative. Conversely, an excess of CAP rate fragments sleep and impairs its quality. Polysomnographic investigation reveals that untreated insomniac patients exhibit significantly higher values of CAP rate compared to healthy sleepers. Effective hypnotic treatment restores physiological amounts of CAP rate, with specific differences between the administered drugs. The sensitivity of CAP parameters to drug manipulation can provide circumscribed information on the hypnotic properties of active compounds.

Relationship of slow and rapid EEG components of CAP to ASDA arousals in normal sleep.

STUDY OBJECTIVES: Besides arousals (according to the ASDA definition), sleep contains also K-complexes and delta bursts which, in spite of their sleep-like features, are endowed with activating effects on autonomic functions. The link between phasic delta activities and enhancement of vegetative functions indicates the possibility of physiological activation without sleep disruption (i.e., arousal without awakening). A functional connection seems to include slow (K-complexes and delta bursts) and rapid (arousals) EEG events within the comprehensive term of activating complexes. CAP (cyclic alternating pattern) is the spontaneous EEG rhythm that ties both slow and rapid activating complexes together during NREM sleep. The present study aims at exploring the relationship between arousals and CAP components in a selected sample of healthy sleepers. DESIGN: Polysomnographic analysis according to the scoring rules for sleep stages and arousals. CAP analysis included also tabulation of subtypes A1 (slow EEG activating complexes), A2 and A3 (activating complexes with fast EEG components). SETTING: 40 sleep-lab accomplished recordings. PARTICIPANTS: Healthy subjects belonging to a wide age range (38 +/- 20 yrs.). INTERVENTIONS: N/A. MEASUREMENT AND RESULTS: Of all the arousals occurring in NREM sleep, 87% were inserted within CAP. Subtypes A2 and A3 of CAP corresponded strikingly with arousals (r=0.843; p<0.0001), while no statistical relationship emerged when arousals were matched with subtypes A1 of CAP. Subtypes A1 instead correlated positively with the percentages of deep sleep (r=0.366; p<0.02). CONCLUSIONS: The CAP subtype classification encompasses both the process of sleep maintenance (subtypes A1) and sleep fragmentation (subtypes A2 and A3), and provides a periodicity dimension to the activating events of NREM sleep.

Cyclic alternating pattern (CAP) and epilepsy during sleep: how a physiological rhythm modulates a pathological event.

OBJECTIVES: Epileptic susceptibility is triggered by the sleeping condition. However, both ictal and interictal events are not equally affected by the different sleep states. Besides the well-known dichotomy between non-REM sleep (high activation) and REM sleep (low activation), epileptic phenomena are deeply sensitive to the ongoing level of arousal. METHODS: During non-REM sleep the arousal level can be either unstable, as expressed by the repetitive sequences of the cyclic alternating pattern (CAP), or stable, as reflected by non-CAP. Phase A (arousal complex) and phase B (post-arousal rebound response) are the two basic components of the CAP cycle, which presents a 20-40 s periodicity. Three subtypes of A phases can be recognized: the A1 subtypes, which are thoroughly composed of K-complexes and delta bursts, and subtypes A2 and A3 dominated by moderate (A2) or prominent (A3) EEG desynchrony. RESULTS: As a manifestation of unstable sleep, CAP offers a favorable background for the occurrence of nocturnal motor seizures that in most cases arise in concomitance with a phase A. In primary generalized epilepsy (PGE) and in lesional epilepsies with fronto-temporal focus, activation of interictal discharges is high during CAP reaching the climax during phase A and the strongest inhibition during phase B. A lack of modulation is observed instead in epilepsy with benign rolandic spikes. In PGE, the interictal bursts are mostly associated with the highly synchronized phase A1 subtypes. CONCLUSIONS: The analysis of sleep microstructure based on CAP parameters offers a sensitive framework for exploring the linkage between dynamic EEG events and epileptic phenomena.

Sleep reactivity during acute nasal CPAP in obstructive sleep apnea syndrome.

OBJECTIVE: To measure the readjustments of sleep macro- and microstructure in patients with obstructive sleep apnea syndrome (OSAS) after acute nasal continuous positive airway pressure (NCPAP) treatment. BACKGROUND: The conventional polysomnographic analysis (macrostructure of sleep) does not necessarily provide the best measures of sleep disruption associated with OSAS. In contrast, microstructural methods of analyzing sleep (i.e., arousals and cyclic alternating pattern) may improve evaluation of patients with OSAS. METHOD: - Ten patients with OSAS were monitored polygraphically before and during the first night of NCPAP therapy. The results were compared with those of 10 age- and sex-matched controls without sleep-related breathing disorders. Each nocturnal recording was followed by daytime observation using the multiple sleep latency test and Visual Analogue Scale (VAS). RESULTS: The first night of ventilatory therapy was characterized by a remarkable expansion of stages 3 and 4 and of REM sleep. In addition, NCPAP suppressed the presence of cyclic alternating pattern (CAP) in REM sleep and induced an impressive rebound of arousals and of certain CAP variables-i.e., CAP rate, CAP time, number of CAP cycles-which dropped well below the physiologic values expressed by controls. A normal duration of phases A and B was re-established starting the first treatment night. When we matched sleep variables with the indices of daytime function, a significant correlation emerged only between the variations of CAP rate and VAS scores. In particular, improvement of daytime sleepiness was less evident when the ventilatory-induced drop of CAP rate was more pronounced. CONCLUSIONS: The application of CAP variables to the microstructural analysis of sleep may expand our knowledge regarding sleep and respiration.

Cyclic alternating pattern and spectral analysis of heart rate variability during normal sleep.

The natural arousal rhythm of non-rapid eye movement (NREM) sleep is known as the cyclic alternating pattern (CAP), which consists of arousal-related phasic events (Phase A) that periodically interrupt the tonic theta/delta activities of NREM sleep (Phase B). The complementary condition, i.e. non-CAP (NCAP), consists of a rhythmic electroencephalogram background with few, randomly distributed arousal-related phasic events. Recently, some relation between CAP and autonomic function has been preliminarily reported during sleep in young adults by means of spectral analysis of heart rate variability (HRV). The present study was aimed at analysing the effects of CAP on HRV in a group of normal children and adolescents. Six normal children and adolescents (age range 10.0-17.5 y) were included in this study. All-night polygraphic recordings were performed after adaptation to the sleep laboratory. Six 5-min epochs were selected from sleep Stage 2 and six from Stages 3 and 4 (slow-wave sleep), both in CAP and NCAP conditions. From such epochs, a series of parameters describing HRV was then calculated, in both time and frequency domains, on the electrocardiographic R-R intervals. Statistical comparison between CAP and NCAP epochs revealed a significant difference for most of the frequency domain parameters (increase of the low-frequency band, increase of the low-frequency/high-frequency ratio and decrease in the high-frequency band during CAP) both in Stage 2 and in slow-wave sleep. Our results demonstrate that the physiological fluctuations of arousal during sleep described as CAP are accompanied by subtle, but significant, changes in balance between the sympathetic and vagal components of the autonomic system.

CAP components and EEG synchronization in the first 3 sleep cycles.

OBJECTIVE: There is consolidated evidence that stage changes in sleep are closely related to spontaneous EEG fluctuations centered on the 20-40 periodicity of the cyclic alternating pattern (CAP). The present investigation aimed at assessing the involvement of the different components of CAP in the process of build-up, maintenance and demolition of deep non-REM (NREM) sleep. METHODS: CAP parameters were quantified in the first 3 sleep cycles (SC1, SC2, SC3), selected from polysomnographic recordings of 25 healthy sound sleepers belonging to an extensive age range (10-49 years). Only ideal SCs were selected, i.e. the ones uninterrupted by intervening wakefulness and in which all stages were represented and linked in a regular succession of a descending branch, a trough and an ascending branch. RESULTS: Among the first 3 SCs, a total amount of 45 (SC1, 16; SC2, 13; SC3, 16) met the inclusion requirements. SCI contained the highest amount of slow wave sleep (43.7 min) and the lowest values of CAP rate (31.6%). The number of phase A1 subtypes remained unmodified across the 3 SCs (SC1, 48; SC2, 48; SC3, 48), whereas both subtypes A2 (SC1, 9; SC2, 14; SC3, 14) and A3 (SC1, 2; SC2, 8; SC3, 10) increased significantly (P<0.028 and P<0.0001, respectively). The A1 subtypes composed more than 90% of all the A phases collected in the descending branches and in the troughs, while the A2 and A3 subtypes were the major representatives (64.3%) of the A phases occurring in the ascending branches. CONCLUSIONS: Within the dynamic organization of sleep, the non-random distribution of CAP sequences, with their succession of slow (subtypes A1) and rapid (subtypes A2 and A3) EEG shifts, seem to be responsible for sculpturing EEG synchrony under the driving and alternating forces of NREM and REM sleep.

Effect of age on EEG arousals in normal sleep.

EEG arousals were quantified in 40 nocturnal polysomnographic recordings belonging to four age groups (teenagers: 10 to 19 years; young adults: 20 to 39 years; middle-aged: 40 to 59 years; elderly: > or = 60 years). Ten subjects (five males and five females) participated in each group. The subjects were healthy and sound sleepers. All sleep recordings were preceded by an adaptation night which aimed at excluding the presence of sleep-related disorders. The recordings were carried out in a partially soundproof recording chamber and in a standard laboratory setting. Arousal indices (AI), defined as the number of arousals per hour of sleep, were calculated for total sleep time (AI/TST) and for all the sleep stages. AI/TST increased linearly with age (r = 0.852; p < 0.00001): teenagers (13.8), young adults (14.7), middle-aged (17.8), elderly (27.1). An age-related positive linear correlation was found also for the arousal indices referred to NREM sleep (r = 0.811; p < 0.00001) and to stages 1 and 2 (r = 0.712; p < 0.00001), while in stages 3 and 4 and in REM sleep, arousal indices showed stable values across the ages. Overall, arousals lasted 14.9 +/- 2.3 seconds, with arousal duration stable across the ages (range of means: 13.3-16.6 seconds) and no relevant differences between NREM sleep (14.6 +/- 2.5 seconds) and REM sleep (16.2 +/- 5 seconds). The paper discusses the impact of age on arousals, the similarities between arousals and the phases d'activation transitoire, and the consideration that arousals are physiological components of sleep.