Cleaved caspase-3 is present in the majority of glial cells in the intact rat spinal cord during postnatal life.

Cell death is an essential process that occurs during the development of the central nervous system. Despite the availability of a wide range of commercially produced antibodies against various apoptotic markers, data regarding apoptosis in intact spinal cord during postnatal development and adulthood are mostly missing. We investigated apoptosis in rat spinal cord at different stages of ontogenesis (postnatal days 8, 29, and 90). For this purpose, we applied immunofluorescent detection of two widely used apoptotic markers, cleaved caspase-3 (cC3) and cleaved poly(ADP-ribose) polymerase (cPARP). Surprisingly, we found significant discrepancy between the number of cC3+ cells and PARP+ cells, with a ratio between 500:1 and 5000:1 in rat spinal cord at all postnatal time points. The majority of cC3+ cells were glial cells and did not exhibit an apoptotic phenotype. In contrast with in vivo results, in vitro analysis of primary cell cultures derived from neonatal rat spinal cord and treated with the apoptotic inductor staurosporine revealed a similar onset of occurrence of both cC3 and cPARP in cells subjected to apoptosis. Gene expression analysis of spinal cord revealed elevated expression of the Birc4 (XIAP), Birc2, and Birc5 (Survivin) genes, which are known potent inhibitors of apoptosis. Our data indicate that cC3 is not an exclusive marker of apoptosis, especially in glial cells, owing its possible presence in inhibited forms and/or its participation in other non-apoptotic roles. Therefore, cPARP appears to be a more appropriate marker to detect apoptosis.

Strong coincidence between slow wave sleep and low AHI is explainable by the high instability of slow wave sleep to obstructive apnea exposure.

It is well known that in patients with obstructive sleep apnea syndrome (OSAS) the apnea-hypopnea index (AHI) is significantly decreased during slow wave sleep (SWS). It used to be explained by the ability of SWS to stabilize the upper airways against collapse. Another explanation, which is the focus of the current study, is that it is just a result of high instability of SWS to obstructive apnea exposure, i.e. high susceptibility of SWS to transition into lighter sleep stages during exposure to obstructive apneas. A retrospective chart review was performed on 560 males who underwent an overnight polysomnography. Two hundred and eighty-seven patients were eligible for the study. They were divided into 3 groups according to different AHI level. All three groups had a higher SWS occurrence in the lateral position than in the supine position. A special fourth group of patients was created with severe OSAS in the supine position but with very mild OSAS in the lateral position. This group had, in the lateral position, (A) higher AHI in NREM sleep (4.1+/-3.1/h vs. 0.7+/-1.2/h, p<0.001) as well as (B) higher SWS occurrence (27.7+/-15.0 % vs. 21.4+/-16.2 % of NREM sleep, p<0.05), than the group with the lowest AHI in the study, i.e. AHI<5/h in NREM sleep. These data suggest that strong coincidence between SWS and low AHI is the result of the high instability of SWS to obstructive apnea exposure. The data also support the presence of SWS-rebound in OSAS patients in the lateral body position.

Skin wound healing in obese and lean male adolescent rats submitted to pre-weaning litter size manipulation.

We investigated whether early postnatal over-nutrition affects normal course of skin wound healing. To induce over-nutrition the litter size was adjusted on the first day after birth to four pups/nest (small litters). In parallel, as a control, normal nests of 10 pups/nest (normal litters) were used. For the wound healing experiment 30 male Sprague-Dawley rats, 15 from normal nests and 15 from small nests, were used. Two parallel full-thickness skin incisions and two full-thickness excisions were performed on the back of each rat. Samples for histological examination (excisions) and wound tensile strength measurement (incisions) were collected on days 2, 6, and 14 after surgery. Our study demonstrates that rats from the small nests had enhanced plasma levels of insulin and enhanced body weight/fat parameters. Furthermore, in small nests, rats that expressed the above-mentioned symptoms displayed slight improvement of epidermis regeneration, accelerated demarcation line formation, and increased wound tensile strength. From this point of view the small nest model used in the present experiment is helpful for exploration whether these acquired changes might be considered as a sufficient essential factor involved in the regulation of metabolic homeostasis and wound repair in juvenile obese male rats. Nevertheless, further studies need to be performed to verify the present findings also on other animal models and humans and to describe the exact underlying mechanism.

Does obstructive sleep apnea worsen during REM sleep?

Although it is thought that obstructive sleep apnea (OSA) is worse during rapid eye movement (REM) sleep than in non-REM (NREM) sleep there are some uncertainties, especially about apnoe-hypopnoe-index (AHI). Several studies found no significant difference in AHI between both sleep stages. However, REM sleep is associated more with side sleeping compared to NREM sleep, which suggests that body position is a possible confounding factor. The main purpose of this study was to compare the AHI in REM and NREM sleep in both supine and lateral body position. A retrospective study was performed on 422 consecutive patients who underwent an overnight polysomnography. Women had higher AHI in REM sleep than NREM sleep in both supine (46.05+/-26.26 vs. 23.91+/-30.96, P<0.01) and lateral (18.16+/-27.68 vs. 11.30+/-21.09, P<0.01) body position. Men had higher AHI in REM sleep than NREM sleep in lateral body position (28.94+/-28.44 vs. 23.58+/-27.31, P<0.01), however, they did not reach statistical significance in supine position (49.12+/-32.03 in REM sleep vs. 45.78+/-34.02 in NREM sleep, P=0.50). In conclusion, our data suggest that REM sleep is a contributing factor for OSA in women as well as in men, at least in lateral position.

Deep nasal inspirations increase the cough threshold in children with mild asthma.

Asthma is a chronic inflammatory disease characterized by bronchospasms accompanied with frequent coughing, the pathogenesis of which is not clear. In healthy adults deep inspirations (DIs) provide a protective effect against bronchoconstriction triggered by methacholine inhalation, which correlates with the number of accompanying cough efforts. The aim was to study the effect of deep nasal inspirations representing the voluntary equivalent of the sniff-like aspiration reflex on the capsaicin-induced cough in children with mild asthma. The cough reflex sensitivity to capsaicin was determined using a compressed air-driven nebulizer in 21 children (8 girls and 13 boys of median age 13.3 year) suffering from mild asthma (FEV(1)∼80%). The effect of five previous DIs through the nose was examined on the elicitability of two and five or more cough efforts (C2, C5). Under control conditions, the concentration of 20.86 (14.58-29.8) µmol/l of capsaicin provoked two cough efforts (C2). After five DIs similar reaction required significantly higher concentrations of capsaicin: 29.02 (18.88-44.6) µmol/l; P=0.016. Five or more cough efforts (C5) were not significantly changed after previous DIs 161.49 (77.31-337.33) µmol/l and without DIs 141.52 (68.77-291); P=0.54. A series of five deep inspirations decreases the cough reflex sensitivity to evoke two efforts (C2) in children with mild asthma. The inhibitory effect of similar DIs disappeared after repeated applications of increasing doses of capsaicin, aiming to evoke five or more cough efforts, suggesting a reflex character of protective effect of DIs.

Severity of nocturnal cardiac arrhythmias correlates with intensity of sleep apnea in men.

Various cardiac arrhythmias frequently occur in patients with sleep apnea, but complex analysis of the relationship between their severity and the probable arrhythmogenic risk factors is conflicting. The question is what cardiovascular risk factors and how strongly they are associated with the severity of cardiac arrhythmias in sleep apnea. Adult males (33 with and 16 without sleep apnea), matched for cardiovascular co-morbidity were studied by polysomnography with simultaneous ECG monitoring. Arrhythmia severity was evaluated for each subject by a special 7-degree scoring system. Laboratory, clinical, echocardiographic, carotid ultrasonographic, ambulatory blood pressure, and baroreflex sensitivity values were also assessed. Moderate sleep apnea patients had benign, but more exaggerated cardiac arrhythmias than control subjects (2.53 ± 2.49 vs. 1.13 ± 1.64 degrees of cumulative severity, p < 0.05). We confirmed strong correlations between the arrhythmia severity and known arrhythmogenic risk factors (left ventricular ejection fraction and dimensions, right ventricular diameter, baroreflex sensitivity, carotid intima-media thickness, age, previous myocardial infarction, and also apnea-hypopnea index). In multivariate modelling only the apnea-hypopnea index indicating the sleep apnea intensity remained highly significantly correlated with the cumulative arrhythmia severity (beta = 0.548, p < 0.005). In conclusion, sleep apnea modifying cardiovascular risk factors and structures or functions provoked various nocturnal arrhythmias. The proposed scoring system allowed a complex analysis of the contribution of various triggers to arrhythmogenesis and confirmed the apnea-hypopnea index as an independent risk for nocturnal cardiac arrhythmia severity in sleep apnea.

Eye tracking using artificial neural networks for human computer interaction.

This paper describes an ongoing project that has the aim to develop a low cost application to replace a computer mouse for people with physical impairment. The application is based on an eye tracking algorithm and assumes that the camera and the head position are fixed. Color tracking and template matching methods are used for pupil detection. Calibration is provided by neural networks as well as by parametric interpolation methods. Neural networks use back-propagation for learning and bipolar sigmoid function is chosen as the activation function. The user's eye is scanned with a simple web camera with backlight compensation which is attached to a head fixation device. Neural networks significantly outperform parametric interpolation techniques: 1) the calibration procedure is faster as they require less calibration marks and 2) cursor control is more precise. The system in its current stage of development is able to distinguish regions at least on the level of desktop icons. The main limitation of the proposed method is the lack of head-pose invariance and its relative sensitivity to illumination (especially to incidental pupil reflections).

Heart rate variability in hypertension caused by sleep disordered breathing and its modification by CPAP.

OBJECTIVES: 1) To analyze heart rate variability (HRV) changes, reflecting the sympathovagal balance with secondary hypertension caused by sleep disordered breathing (SDB), compared to healthy controls and essential hypertension without SDB; 2) to compare HRV changes between various degrees of SDB severity; and 3) to test the modification of HRV indices by continuous positive airway pressure (CPAP) in SDB patients. BACKGROUND: Differentiation of secondary hypertension caused by SDB from essential hypertension and healthy controls by ambulatory blood pressure measurement (ABPM) and its modification by CPAP, requires an analysis of HRV changes, as frequently used for the prediction of cardiovascular risk. METHODS: HRV changes were analyzed in 48 adults divided into six groups according to the apnoea/hypopnoea index (AHI), i.e. three groups with various degrees of SDB, a group with severe SDB after CPAP application, a group with essential hypertension without SDB, and a group of healthy controls. Night-time and daytime values of low frequency (LF) and high frequency (HF) bands and the LF/HF ratio were compared in the six groups. RESULTS: The night-time values of LF bands were higher in severe than in moderate and mild degrees of SDB, and the correlation of LF/HF ratio with AHI (r = 0.3511) suggests the gradual increase of sympathetic predominance with the severity of SDB. The high sympathetic activity substantially decreased after application of CPAP in severe SDB. CONCLUSION: The increased nocturnal values of the LF band and the LF/HF ratio, caused by frequent apnoea/ hypopnoea episodes, support the usefulness of HRV spectral analysis for the prediction of cardiovascular risk in patients with SDB (Tab. 1, Fig. 3, Ref. 36).

Management of obstructive sleep apnea in Europe.

OBJECTIVES: In Europe, the services provided for the investigation and management of obstructive sleep apnoea (OSA) varies from country to country. The aim of this questionnaire-based study was to investigate the current status of diagnostic pathways and therapeutic approaches applied in the treatment of OSA in Europe, qualification requirements of physicians involved in diagnosis and treatment of OSA, and reimbursement of these services. METHODS: Two questionnaires were sent to 39 physicians in 22 countries in Europe. In order to standardize the responses, the questionnaire was accompanied by an example. RESULTS: Sleep centers from 21 countries (38 physicians) participated. A broad consistency among countries with respect to the following was found: pathways included referral to sleep physicians/sleep laboratories, necessity for objective diagnosis (primarily by polysomnography), use of polygraphic methods, analysis of polysomnography (PSG), indications for positive airway pressure (PAP) therapy, application of standard continuous PAP (CPAP) therapy (100% with an CPAP/APAP ratio of 2.24:1), and the need (90.5%) and management of follow-up. Differences were apparent in reimbursement of the diagnostic procedures and follow-up, in the procedures for PAP titration from home APAP titration with portable sleep apnea monitoring (38.1%) up to hospital monitoring with PSG and APAP (85.7%), and in the qualification requirements of sleep physicians. CONCLUSIONS: Management of OSA in different European countries is similar except for reimbursement rules, qualification of sleep specialists and procedures for titration of the CPAP treatment. A European network (such as the one accomplished by the European Cooperation in Science and Technology [COST] B26 Action) could be helpful for implementing these findings into health-service research in order to standardize management in a cost effective perspective.

Intradermal running suture versus 3M™ Vetbond™ tissue adhesive for wound closure in rodents: a biomechanical and histological study.

BACKGROUND: Although tissue adhesives and traditional sutures were compared in numerous studies during the early stages of healing, it has to be clarified, from the histological and biomechanical point of view, how the differences develop during the later phases. METHODS: Twenty-four male Sprague-Dawley rats were used and divided into 2 groups: the IRS (intradermal running suture) and G (glue) groups. Two parallel full-thickness skin incisions were made on the back of each rat. Wounds in the IRS group were closed by an IRS, whereas wounds in the G group were closed using tissue adhesive (n-butylcyanoacrylate). Rats were sacrificed 7 and 22 days after surgery. RESULTS: Similar wound tensile strengths of glued and sutured wounds were measured on days 7 (IRS = 10.3 ± 1.7 g/mm(2) vs. G = 12.9 ± 4.0 g/mm(2), p = 0.9612) and 22 (IRS = 95.6 ± 15.7 g/mm(2) vs. G = 85.6 ± 16.4 g/mm(2), p = 0.2502) after surgery. Histology revealed a significantly increased amount of granulation tissue formation in glued wounds on day 7. The difference in granulation tissue formation was reduced until day 22. CONCLUSIONS: Tissue adhesive based on n-butylcyanoacrylate presents a fair alternative to traditional suture wound closure techniques.

Distinct generators for aspiration and expiration reflexes: localization, mechanisms and effects.

Re-evaluation of our earlier c-Fos-like immuno-reactive studies and brainstem transection/lesion experiments in over 40 anaesthetized, non-paralyzed cats allowed comparison of two distinct airway defensive reflexes with the distinct generators for inspiration (I) and expiration (E), described recently in juvenile rats. The spiration reflex (AspR) is characterized by solitary rapid and strong inspiratory effort with a reciprocal inhibition, preventing a subsequent active expiration, while the expiration reflex (ExpR) manifests by rapid and strong expiratory effort, starting without a preceding, inspiration, or reciprocal inhibition of occasional spontaneous inspiration. The retro-trapezoid nucleus/parafacial respiratory group neurones described as the distinct generator for active E in rats, are activated also during the ExpR in adult cats. Brainstem transection 5 mm above the obex eliminates the E generator and the ExpR, but preserves the I generator located in the pre-Bötzinger Complex, and also the AspR. This suggests the existence of a distinct I generator in cats as well as rats, and its contribution to the generation of the AspR. Persistence of the AspR in adult cats during asphyxic gasping, their similar character and the strong activation of I neurones at many places in the medulla and pons, suggest a common brainstem neuronal circuit contributing to generation of both the gasping and the gasp-like AspR. That the AspR and ExpR have distinct multilevel brainstem control mechanisms supports the dual theory of control and provides unique models for testing respiratory rhythm and pattern generation. The AspR may be compared with the powerful "auto-resuscitation effects of asphyxic gasping"; the ExpR may underly the effectiveness of the laryngeal chemoreflexes in prevention of lung diseases.

Provocation of aspiration reflexes and their effects on the pattern of cough and reflex apnea in cats.

Aspiration reflexes (AspRs) manifesting as reflex spasmodic inspirations and their effects on motor pattern of tracheobronchial cough and reflex apnea were studied on 22 spontaneously breathing pentobarbitone-anesthetized cats. AspRs induced during cough inspiration enhanced peak inspiratory (P<0.01) and expiratory (P<0.02) esophageal pressures, amplitudes of diaphragm (P<0.01) and abdominal muscles (P<0.05) EMG activity, and prolonged the entire expiratory period (P<0.01) and total cycle duration (P<0.05) of cough. Transient inhibitions and splits of cough expiration frequently occurred with AspR within active cough expiratory period; however, cough spatiotemporal characteristics were not altered significantly. Sub-threshold nasopharyngeal stimulation failing to provoke AspR had no significant effects on coughing. Hering-Breuer inflation apnea was moderately prolonged by AspRs (20%; P<0.05), unlike the apnea produced by continual mechanical laryngeal stimulation. AspRs are inducible during tested behaviors interacting with their motor pattern. Central mechanisms involving pulmonary stretch receptor stimulation is suggested for modulation of cough and inflation apnea by AspR.

Excitability and rhythmicity of tracheobronchial cough is altered by aspiration reflex in cats.

Effects of nasopharyngeal stimulation on excitability and rhythmicity of mechanically induced tracheobronchial cough were examined on 18 pentobarbitone anesthetized cats. After the 17.2+/-2.4 aspiration reflexes (AspRs), tracheobronchial stimulation evoked lower number of coughs (P<0.05) with longer latency to the diaphragm activation (P<0.02), compared to control. AspRs induced within "inter-cough" periods (motor quiescence between individual coughs; 3.3 AspRs per period) reduced cough number by 50% (P<0.01), shortened cough-related abdominal activity (P<0.02), prolonged inter-cough period (P<0.02), and the total cough cycle duration (P<0.05). Cough efforts occurred irregularly with very variable "inter-cough" distances and total cough cycle durations. The subthreshold nasopharyngeal stimulation (failing to evoke AspR) did not affect rhythmic coughing. AspRs induced during persisting post-stimulation coughs did not significantly reduce their number. Excitability and rhythmicity of mechanically induced tracheobronchial cough can be reduced by AspRs, but not by subthreshold nasopharyngeal stimulation. The suppressive effect of spasmodic inspirations on chronic cough is suggested.

[Traditional risk factors of atherosclerosis in patients with obstructive sleep apnoe-hypopnoe syndrome]. / Tradicné rizikové faktory aterosklerózy u pacientov so syndrómom obstrukcného spánkového apnoe-hypopnoe.

UNLABELLED: Cardiovascular morbidity and mortality of patients with obstructive sleep apnoe-hypopnoe syndrome (OSAHS) is higher than in matched population. The aim of the study was to analyse, whether high prevalence of risk factors of atherosclerosis in patients with OSAHS can explaine their higher cardiovascular morbidity. METHODS: 2 groups of 33 males with moderate OSAHS and 16 males without OSAHS were subdivided into subgroups of apparently healthy subjects, hypertonics and patients with coronary artery disease. We compared the presence of traditional risk factors of atherosclerosis (age, hyperlipoproteinaemia, diabetes mellitus, arterial hypertension, obesity, smoking habit, obesity, family history), compensation of metabolic risk factors, morphometry of the heart, haemodynamic parameters, markers of atherosclerosis, microalbuminuria and some respiratory parameters. RESULTS: Patients with/without OSAHS had a comparable risk profile at all grades of cardiovascular pathology, they differed only in respiratory parameters (characteristic for OSAHS). Moreover, OSAHS patients without cardiovascular diseases vs. matched non OSAHS subjects had higher thickness of posterior wall of the left ventricule (9.73 +/- 1.17 vs 8.29 +/- 1.38 mm, p < 0.04), intima-media thickness of carotid artery (0.83 +/- 0.14 vs 0.63 +/- 0.08 mm, p < 0.001) and OSAHS patients with hypertension vs non OSAHS hypertonics presented higher dimension of right ventricule (28.4 +/- 2.7 vs 26.25 +/- 0.96 mm, p < 0.02), and left ventricule (47.6 +/- 3.3 vs 43.75 +/- 0.96 mm, p < 0.01). CONCLUSION: Patients with OSAHS had severe atherosclerosis and worse morphological alteration of the heart compared to subjects without OSAHS. According to comparable presence and possibility of sufficient control of risk factors, OSAHS can be considered to be a risk factor for cardiovascular diseases. However, this statement has to be further confirmed in prospective studies.

Medico-legal implications of sleep apnoea syndrome: driving license regulations in Europe.

BACKGROUND: Sleep apnoea syndrome (SAS), one of the main medical causes of excessive daytime sleepiness, has been shown to be a risk factor for traffic accidents. Treating SAS results in a normalized rate of traffic accidents. As part of the COST Action B-26, we looked at driving license regulations, and especially at its medical aspects in the European region. METHODS: We obtained data from Transport Authorities in 25 countries (Austria, AT; Belgium, BE; Czech Republic, CZ; Denmark, DK; Estonia, EE; Finland, FI; France, FR; Germany, DE; Greece, GR; Hungary, HU; Ireland, IE; Italy, IT; Lithuania, LT; Luxembourg, LU; Malta, MT; Netherlands, NL; Norway, EC; Poland, PL; Portugal, PT; Slovakia, SK; Slovenia, SI; Spain, ES; Sweden, SE; Switzerland, CH; United Kingdom, UK). RESULTS: Driving license regulations date from 1997 onwards. Excessive daytime sleepiness is mentioned in nine, whereas sleep apnoea syndrome is mentioned in 10 countries. A patient with untreated sleep apnoea is always considered unfit to drive. To recover the driving capacity, seven countries rely on a physician's medical certificate based on symptom control and compliance with therapy, whereas in two countries it is up to the patient to decide (on his doctor's advice) to drive again. Only FR requires a normalized electroencephalography (EEG)-based Maintenance of Wakefulness Test for professional drivers. Rare conditions (e.g., narcolepsy) are considered a driving safety risk more frequently than sleep apnoea syndrome. CONCLUSION: Despite the available scientific evidence, most countries in Europe do not include sleep apnoea syndrome or excessive daytime sleepiness among the specific medical conditions to be considered when judging whether or not a person is fit to drive. A unified European Directive seems desirable.

Sleep apnoea inducing hypoxemia is associated with early signs of carotid atherosclerosis in males.

The intima-media thickness (IMT) of carotid arteries as a marker of preclinical atherosclerosis was measured by ultrasonography in 49 subjects to determine, how strongly the obstructive sleep apnoea (OSA) syndrome is associated with atherosclerosis. Maximal IMT was higher in patients with cardiovascular diseases and with or without risk factors of atherosclerosis, presenting also OSA (apnoea-hypopnoea index=26.1+/-15.6/h) compared to controls without OSA (0.91+/-0.21 mm versus 0.77+/-0.18 mm, p<0.05). The prevalence of IMT > or = 0.85 mm was also higher in patients with cardiovascular pathology presenting OSA than without it (p<0.05). IMT(max) was increased in subjects with mild to moderate OSA alone (AHI=20.4+/-8.7/h) versus healthy controls (0.83+/-0.14 mm versus 0.63+/-0.08 mm, p<0.01). Regression analysis revealed a correlation of IMT(max) with the frequency, intensity and duration of intermittent hypoxemia reflected by AHI (p<0.01), minimal oxygen saturation (p<0.01) and time spent with Sa(O2) < 90% (p<0.05) in patients presenting OSA. The results indicate clear association between early signs of carotid atherosclerosis and moderate OSA in males with and without concomitant cardiovascular pathology.

Development of GABA-sensitive spasticity and rigidity in rats after transient spinal cord ischemia: a qualitative and quantitative electrophysiological and histopathological study.

Transient spinal cord ischemia may lead to a progressive degeneration of spinal interneurons and subsequently to increased hind limb motor tone. In the present work we sought to characterize the rigidity and spasticity components of this altered motor function by: i) tonic electromyographic activity measured in gastrocnemius muscle before and after ischemia, ii) measurement of muscle resistance during the period of ankle flexion and corresponding changes in electromyographic activity, iii) changes in Hoffmann reflex, and, iv) motor evoked potentials. In addition the effect of intrathecal treatment with baclofen (GABAB receptor agonist; 1 microg), nipecotic acid (GABA uptake inhibitor; 300 microg) and dorsal L2-L5 rhizotomy on spasticity and rigidity was studied. Finally, the changes in spinal choline acetyltransferase (ChAT) and vesicular glutamate transporter 2 and 1 (VGLUT2 and VGLUT1) expression were characterized using immunofluorescence and confocal microscopy. At 3-7 days after ischemia an increase in tonic electromyographic activity with a variable degree of rigidity was seen. In animals with modest rigidity a velocity-dependent increase in muscle resistance and corresponding appearance in electromyographic activity (consistent with the presence of spasticity) was measured during ankle rotation (4-612 degrees /s rotation). Measurement of the H-reflex revealed a significant increase in Hmax/Mmax ratio and a significant loss of rate-dependent inhibition. In the same animals a potent increase in motor evoked potential amplitudes was measured and this change correlated positively with the increased H-reflex responses. Spasticity and rigidity were consistently present for a minimum of 3 months after ischemia. Intrathecal treatment with baclofen (GABA B receptor agonist) and nipecotic acid (GABA uptake inhibitor) provided a significant suppression of spasticity, rigidity, H-reflex or motor evoked potentials. Dorsal L2-L5 rhizotomy significantly decreased muscle resistance but had no effect on increased amplitudes of motor evoked potentials. Confocal analysis of spinal cord sections at 8 weeks-12 months after ischemia revealed a continuing presence of ChAT positive alpha-motoneurons, Ia afferents and VGLUT2 and VGLUT1-positive terminals but a selective loss of small presumably inhibitory interneurons between laminae V-VII. These data demonstrate that brief transient spinal cord ischemia in rat leads to a consistent development of spasticity and rigidity. The lack of significant suppressive effect of dorsal L2-L5 rhizotomy on motor evoked potentials response indicates that descending motor input into alpha-motoneurons is independent on Ia afferent couplings and can independently contribute to increased alpha-motoneuronal excitability. The pharmacology of this effect emphasizes the potent role of GABAergic type B receptors in regulating both the spasticity and rigidity.

Cough and laryngeal muscle discharges in brainstem lesioned anaesthetized cats.

Experiments were carried out to determine whether there are separate drives from the selected neuronal networks of the brainstem affecting the discharge patterns of laryngeal and respiratory pump muscles during cough. Twenty-four non-decerebrate spontaneously breathing cats anesthetized with sodium pentobarbitone were used. Microinjections of kainic acid into the lateral tegmental field of the medulla, medullary midline or pontine respiratory group eliminated the cough evoked by mechanical stimulation of the tracheobronchial and laryngopharyngeal mucosa. These stimuli, in most cases, provoked irregular bursts of discharges in the posterior cricoarytenoid and thyroarytenoid laryngeal muscles (or they had no effect on them). No pattern of laryngeal muscle activities following lesions resembled the laryngeal cough response. Lesions of the target regions did not result in any apparent changes in the eupnoeic pattern of laryngeal activity. Neurons of the medullary lateral tegmental field, raphe nuclei and the pontine respiratory group seem to be indispensable for the configuration of the central cough motor pattern. However, these neurons do not appear to be essential for the discharge patterns of laryngeal motoneurons during eupnoea. The residual laryngeal "cough" responses are probably mediated by an additional motor drive.

Interactive measurement and characterization of DNA molecules by analysis of AFM images.

BACKGROUND: In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. METHODS: Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. RESULTS: The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. CONCLUSIONS: An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.

Brainstem areas involved in the aspiration reflex: c-Fos study in anesthetized cats.

Expression of the immediate-early gene c-fos, a marker of neuronal activation was employed in adult anesthetized non-decerebrate cats, in order to localize the brainstem neuronal populations functionally related to sniff-like (gasp-like) aspiration reflex (AR). Tissues were immunoprocessed using an antibody raised against amino acids of Fos and the avidin-biotin peroxidase complex method. The level of Fos-like immunoreactivity (FLI) was identified and counted in particular brainstem sections under light microscopy using PC software evaluations in control, unstimulated cats and in cats where the AR was elicited by repeated mechanical stimulation of the nasopharyngeal region. Fourteen brainstem regions with FLI labeling, including thirty-seven nuclei were compared for the number of labeled cells. Compared to the control, a significantly enhanced FLI was determined bilaterally in animals with the AR, at various medullary levels. The areas included the nuclei of the solitary tract (especially the dorsal, interstitial and ventrolateral subnuclei), the ventromedial part of the parvocellular tegmental field (FTL -- lateral nuclei of reticular formation), the lateral reticular nucleus, the ambigual and para-ambigual regions, and the retrofacial nucleus. FLI was also observed in the gigantocellular tegmental field (FTG -- medial nuclei of reticular formation), the spinal trigeminal nucleus, in the medullar raphe nuclei (ncl. raphealis magnus and parvus), and in the medial and lateral vestibular nuclei. Within the pons, a significant FLI was observed bilaterally in the parabrachial nucleus (especially in its lateral subnucleus), the Kolliker-Fuse nucleus, the nucleus coeruleus, within the medial region of brachium conjunctivum, in the ventrolateral part of the pontine FTG and the FTL. Within the mesencephalon a significantly enhanced FLI was found at the central tegmental field (area ventralis tegmenti Tsai), bilaterally. Positive FLI found in columns extending from the caudal medulla oblongata, through the pons up to the mid-mesencephalon suggests that the aspiration reflex is thus co-ordinated by a long loop of medullary-pontine-mesencephalic control circuit rather than by a unique "center".