Clonidine abolishes the adverse effects on apoptosis and behaviour after neonatal ketamine exposure in mice.

BACKGROUND: An increasing amount of both experimental and epidemiological data indicates that neonatal anaesthesia causes disruption of normal brain development in rodents and primates, as manifested by acute increased apoptosis and long-lasting altered behaviour and learning. It is necessary to seek strategies that avoid the possible adverse effects after anaesthesia. Our purpose is to show that increased apoptosis and behavioural alterations after ketamine exposure during this period may be prevented by clonidine, a compound already used by paediatric anaesthetists for sedation. METHODS: To investigate the protective properties of clonidine pre-treatment, five groups of 10-day-old mice were injected with either ketamine 50 mg/kg, clonidine 40 µg/kg, ketamine 50 mg/kg 30 min after 10 µg/kg clonidine, ketamine 50 mg/kg 30 min after 40 µg/kg clonidine or saline (control). Apoptosis was measured 24 h after treatment using Flouro-Jade staining. Spontaneous activity in a novel environment was tested at an age of 55 days. RESULTS: Pre-treatment with 40 µg/kg clonidine, but not 10 µg/kg clonidine, 30 min before ketamine exposure abolished ketamine-induced apoptosis and the behavioural changes observed in the young adult mice. The mice exposed to clonidine alone showed no differences from the saline-treated (control) mice. CONCLUSION: The administration of clonidine eliminated the adverse effects of ketamine in this mouse model, suggesting a possible strategy for protection. Alone, clonidine did not cause any adverse effects in these tests.

Inadequate prophylactic effect of low-molecular weight heparin in critically ill COVID-19 patients.

PURPOSE: The aim of this study was to investigate potential markers of coagulopathy and the effects of thromboprophylaxis with low-molecular-weight heparin (LMWH) on thromboelastography (TEG) and anti-factor Xa in critically ill COVID-19 patients. MATERIAL AND METHODS: We conducted a prospective study in 31 consecutive adult intensive care unit (ICU) patients. TEG with and without heparinase and anti-factor Xa analysis were performed. Standard thromboprophylaxis was given with dalteparin (75-100 IU/kg subcutaneously). RESULTS: Five patients (16%) had symptomatic thromboembolic events. All patients had a maximum amplitude (MA) > 65 mm and 13 (42%) had MA > 72 mm at some point during ICU stay. Anti-factor Xa activity were below the target range in 23% of the patients and above target range in 46% of patients. There was no significant correlation between dalteparin dose and anti-factor Xa activity. CONCLUSIONS: Patients with COVID-19 have hypercoagulability with high MA on TEG. The effect of LMWH on thromboembolic disease, anti-factor Xa activity and TEG was variable and could not be reliably predicted. This indicates that standard prophylactic doses of LMWH may be insufficient. Monitoring coagulation and the LMWH effect is important in patients with COVID-19 but interpreting the results in relation to risk of thromboembolic disease poses difficulties.

Decreased capillarization and a shift to fast myosin heavy chain IIx in the biceps brachii muscle from young adults with spastic paresis.

Muscle spasticity and paresis are conditions that occur secondary to upper motor neuron lesions. The co-existence of decreased motor unit recruitment and intermittent over-activity generates confusion concerning the effect on muscle fiber characteristics. In order to increase the knowledge about the effect of upper motor lesion on capillarization and muscle fiber composition, the biceps brachii muscle from seven young adults with long duration of spastic paresis and seven age-matched controls were analyzed using morphological and enzyme- and immuno-histochemical techniques. The spastic muscles had a 38% lower capillary density (p=0.002), 30% fewer capillaries around each muscle fiber (p=0.02), and 16% fewer capillaries when related to the fiber size (p=0.04). The frequency of fibers expressing myosin heavy chain (MyHC) IIx increased (30% vs. 4%, p=0.006), while the percentage of fibers expressing MyHC I and MyHC IIa, respectively, decreased (22% vs. 46% and 7% vs. 29%, p<0.01). The high proportion of muscle fibers with low oxidative capacity and low capillary supply indicates that biceps brachii muscle from patients with upper motor lesions fatigue more easily than normal controls. We also observed a significantly higher variability in fiber size for fibers expressing MyHC I (p<0.04), and, in three of the subjects, a small amount of small fibers expressing developmental MyHCs was found. These results suggest that, although intermittent stretch reflex contractions might have an impact on the muscle characteristics in spastic paresis, the muscle phenotypic properties are more adapted to decreased voluntary motor unit recruitment.

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction.

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less affected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

Immobilized 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole as solid phase luminophore in peroxyoxalate chemiluminescence.

A new luminophore for application in peroxyoxalate chemiluminescence is presented. An analogue of the well-known chemiluminescence compound lophine, i.e. 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI), has been covalently immobilized to controlled pore glass and a porous methacrylate resin. By using this reagent in a solid phase detection reactor, sensitive determinations of hydrogen peroxide have been demonstrated. In homogeneous solution HCPI emits poorly as a result of 1,1'-oxalyldiimidazole excitation, but when immobilized its efficiency is almost comparable to highly efficient luminophores such as 3-aminofluoranthene. Linearity extends in the single stream flow system over several orders of magnitude with both materials. The limit of detection was 1 nmol/l (10 fmole injected), when using the porous methacrylate support.

Influence of Imidazole and Bis(trichlorophenyl) Oxalate in the Oxalyldiimidazole Peroxyoxalate Chemiluminescence Reaction.

The complex role of imidazole when used as a catalyst in the bis(2,4,6-trichlorophenyl) oxalate (TCPO) peroxyoxalate chemiluminescence (PO-CL) reaction is explained by the transient formation and subsequent degradation of 1,1'-oxalyldiimidazole (ODI). When ODI was used directly as PO-CL reagent, the stability was improved by addition of TCPO as an "imidazole sponge", since ODI is rapidly decomposed in the presence of imidazole. In this way, the imidazole-catalyzed degradation of ODI was hindered efficiently. The stability of ODI was also influenced by the storage vessel material. Polymeric bottles were found to be more suitable than glass containers. A comparison was made between the traditionally used reagent TCPO/imidazole (mixed on-line for formation of ODI) and the new reagent combination ODI-TCPO (premixed) with respect to sensitivity, noise, and background.

Study of isolated mitochondria incubated with labelled and non-labelled alloxan, with regard to intramitochondrial concentrations of reduced glutathione and inorganic phosphate.

Isotope technique and determination of the intramitochondrial concentration of GSH were used with the aim of studying the uptake of alloxan in isolated mouse mitochondria. A significantly decreased concentration of GSH was observed in mitochondria from liver and pancreas incubated with alloxan. This effect was significantly more marked in mitochondria depleted of Pi. Increased radioactivity was found in isolated mitochondria from liver, exocrine pancreas and endocrine pancreas incubated with 14C-2-alloxan. Even this effect was significantly more marked in Pi depleted mitochondria. Isolated liver mitochondria of the KsJ-strain of C57BL-mice possessed a significantly lower concentration of endogenous Pi than those of the 6J-strain. The findings support the view that alloxan can pass across the inner mitochondrial membrane, and suggest that the intramitochondrial concentration of Pi is one of the factors which has an influence on the uptake of alloxan in isolated mouse mitochondria.

Hemodynamic effects of prenalterol, a beta1-adrenoreceptor agonist, in hypotension induced by high thoracic epidural block in man.

The hemodynamic effects of prenalterol, a new beta1-adrenoreceptor agonist, on hypotension induced by a thoracic epidural block extending between T1 and T12 and thereby blocking the cardiac sympathetic supply have been studied in eight patients scheduled for abdominal aortic aneurysm resection. The thoracic epidural block induced a drop in blood pressure, due to a reduction in cardiac output and systemic vascular resistance. Intravenous infusion of 10 mg of prenalterol rapidly and effectively reversed the hypotension by an increase in cardiac output without any effects on systemic vascular resistance or heart rate. The results indicate that prenalterol is a pure beta1 agonist with a marked inotropic but no chronotropic property.

Effect of muscle tension during tendon transfer on sarcomerogenesis in a rabbit model.

Sarcomere number change was investigated in an animal model of tendon transfer. In 9 adult New Zealand white rabbits, the flexor digitorum longus muscle was cut distally and transferred and woven into the tibialis anterior tendon. Ankles were then immobilized for 3 weeks in 75 degrees flexion. Transferred flexor digitorum longus muscles were harvested and complete architectural analysis was performed. Sarcomere lengths were measured using laser diffraction. Serial sarcomere number in transferred flexor digitorum longus fibers was a strong function of the sarcomere length at the time of transfer. A highly significant negative correlation between these 2 parameters was approximated by a linear relationship. Based on this finding, we conclude that serial sarcomere number is significantly affected by the degree of stretch during the transfer itself. This could easily compromise the purpose of surgical tendon transfer by reducing the procedure to little more than a tenodesis. (J Hand Surg 2000; 25A:138-143.

Derivatization of steroids with dansylhydrazine using trifluoromethanesulfonic Acid as catalyst.

A new dansylation reaction, where trifluoromethanesulfonic acid is used as catalyst, has been characterized for six ketosteroids by employing experimental design followed by multivariate data analysis. The molar ratio between the steroid and the derivatization reagent was found to be the factor most strongly affecting the reaction. Faster reaction kinetics was achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the dual peaks observed when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti hydrazones formed. We furthermore conclude that the dansylation reaction is subject to alkyl catalysis rather than acid catalysis, since methyl trifluoromethanesulfonate showed a strong catalytic action, while the catalytic action of trifluoromethanesulfonic acid was lower when diluted in other alcohols and disappeared in aprotic solvents. A sensitivity to water in the reaction mixture strengthens the evidence for alkyl catalysis. When optimal experimental conditions were used, derivatization of picomole amounts of ketosteroids could be accomplished in 25 min. Analysis of spiked plasma containing 0.2-2.0 nmol each of progesterone and 3α-hydroxy-5ß-pregnan-20-one showed overall recoveries of 69-76% and 40-55%, respectively. The corresponding 3σ detection limits estimated from calibration curve data were 12 and 15 pmol (n = 4, 500 µL injected).

Effects of thoracic epidural block and the beta-1-adrenoreceptor agonist prenalterol on the cardiovascular response to infrarenal aortic cross-clamping in man.

Sixteen patients scheduled for abdominal aortic resection and grafting were randomly assigned to two groups to study the cardiovascular effects of infrarenal aortic cross-clamping. The patients in the first group had received a thoracic epidural block followed by intravenous administration of the selective beta-1-adrenoreceptor agonist prenalterol prior to induction of general anaesthesia. The patients in the second group served as controls and received no specific treatment prior to general anaesthesia. In both groups, aortic cross-clamping was followed by an equal rise in pulmonary artery diastolic pressure and mean systemic arterial pressure. There was a significant difference in systemic vascular resistance, as the control group had a 46% increase 30 s after cross-clamping, while the pretreated patients had only a 7% increase at the same time. Moreover, the patients given the thoracic epidural block followed by prenalterol increased their stroke volume and cardiac indices, as compared to the patients in the control group who showed a significant decrease in these parameters. Possible mechanisms for the mode of action of the combined thoracic epidural block and beta-1-adrenoreceptor agonist pretreatment are discussed.

Sarcomere length changes after flexor carpi ulnaris to extensor digitorum communis tendon transfer.

Sarcomere length was measured intraoperatively on five patients undergoing tendon transfer of the flexor carpi ulnaris (FCU) to the extensor digitorum communis (EDC) for radial nerve palsy. The most significant result was that the absolute sarcomere length and sarcomere length operating range of the FCU increased after transfer into the EDC (p < .001). Preoperatively, with the wrist fully extended and fingers flexed, FCU sarcomere length was 4.22 +/- .24 microns and decreased to 3.19 +/- .05 microns as the wrist was fully flexed. This represented an overall sarcomere length range of 1.03 microns. After the tendon transfer using standard recommended techniques, all sarcomere lengths were significantly longer (p < .001). Specifically, sarcomeres were 0.74 +/- .14 microns longer with the muscle in its fully lengthened position (4.96 +/- .43 microns with the wrist and digits flexed) and 0.31 +/- .16 microns longer with the FCU in the fully shortened position (3.50 +/- .06 microns with the wrist and digits extended). At these sarcomere lengths, the FCU muscle was predicted to develop relatively high force only during movement involving synergistic wrist flexion and finger extension. Under the conditions of the procedures performed, the transferred FCU muscle was predicted to produce maximum force over the range of about 30 degrees of wrist flexion and 0 degree of finger flexion to 70 degrees of wrist extension and 90 degrees of finger flexion. While this is acceptable, a more desirable result was predicted to occur if the muscle was transferred at a longer length. In this latter case, greater stretch of the FCU during transfer (increasing sarcomere length to about 5 microns) was predicted to improve the transfer. The more highly stretched FCU was predicted to result in maximum force as the wrist and fingers progressed from about 60 degrees of wrist extension and 0 degree of finger flexion to 80 degrees of wrist extension and 70 degrees of finger flexion. These results quantify the relationship between the passive tension chosen for transfer, sarcomere length, and the estimated active tension that can be generated by the muscle. The results also demonstrate the feasibility of using intraoperative laser diffraction during tendon transfer as a guide for optimal placement of the transferred muscle.

Perfluorosulfonated ionomer-modified polyethylene. A material for simultaneous solid-phase enrichment and enhanced precolumn dansylation of C-21 ketosteroids in human serum.

A new derivatization procedure has been developed where solid-phase catalysis is utilized to facilitate the formation of hydrazones in precolumn labeling of keto-containing compounds. This procedure has been implemented on a solid-phase enrichment and enhanced derivatization (SPEED) device, prepared from porous polyethylene that has been coated with Nafion and dansylhydrazine. The SPEED devices have been optimized using experimental design and characterized for dansylation of C-21 ketosteroids by multivariate data analysis, using progesterone as the model compound. The reaction temperature and the molar ratio between the steroid and the derivatization reagent were found to be the factors most strongly affecting the reaction. Faster reaction kinetics were achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the four derivative peaks eluting when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti mono- and bis(hydrazones) formed in the reaction. Using optimal reaction conditions, the derivatives mainly constitute the syn and anti conformers of bis-derivatives. In contrast to solution-based acid catalysis, the SPEED device was remarkably insensitive to water in the reaction mixture. A sample volume of 400 microL was found to be the maximum, enabling sample enrichment prior derivatization. Using optimal experimental conditions, picomole amounts of ketosteroids could be derivatized in 10 min at room temperature. Analysis of spiked serum samples containing 0.4-2.0 nmol of progesterone showed overall recoveries of 52-63%. The corresponding 3delta detection limit was 1.3 pmol (n = 4, 100 microL injected), as estimated from calibration curve data.