The volume of distribution (Vd) is 1.1 L/kg both in neonates and adults. Midazolam is hydroxylated by CYP3A4 and CYP3A5; the activities of these enzymes surge in the liver in the first weeks of life and thus the metabolic rate of midazolam is lower in neonates than in adults. Midazolam acts as a sedative, as an antiepileptic, for those infants who are refractory to standard antiepileptic therapy, and as an anaesthetic. Information of midazolam as an anaesthetic in infants are very little. Midazolam is usually administered intravenously; when minimal sedation is required, intranasal administration of midazolam is employed. Disease affects the pharmacokinetics of midazolam in neonates; multiple organ failure reduces the Cl of midazolam and mechanical ventilation prolongs the t1/2 of this drug. ECMO therapy increases t1/2, Cl, and Vd of midazolam several times. The adverse effects of midazolam in neonates are scarce: pain, tenderness, and thrombophlebitis may occur. Respiratory depression and hypotension appear in a limited percentage of infants following intravenous infusion of midazolam. In conclusion, midazolam is a safe and effective drug which is employed as a sedative, as antiepileptic agent, for infants who are refractory to standard antiepileptic therapy, and as an anaesthetic.Midazolam is a benzodiazepine with rapid onset of action and short duration of effect. In healthy neonates the half-life ( Midazolam is a short-acting benzodiazepine with rapid onset of action. It has anxiolytic, muscle relaxant, and anticonvulsant activity, now most widely used to generate anterograde amnesia and to stop prolonged seizures in children . MidazolA receptors are responsible for most inhibitory neurotransmission in the central nervous system. Benzodiazepines act at GABAA receptors by binding directly to a specific site that is distinct from that of GABA binding [Midazolam exerts most of its effects by interacting with inhibitory neurotransmitter receptors directly activated by GABA. GABA binding . Midazol binding .t1/2) of midazolam is 1.9 hours, which is 22-fold shorter than that of diazepam [t1/2 of midazolam is shorter in neonates; it is about from 4 to 6 hours. It is quite variable and may be up to 22 hours in premature infants [In adults, the half-life  or with the sedative-hypnotic midazolam in ventilated neonates. Endotracheal intubation and mechanical ventilation are major components of routine intensive care for very low birthweight newborn infants and sick fullterm newborn infants . Sedatioμg/kg per min, with an average of 4 μg/kg per min, terminated the seizures in 32 infants whose seizures could not be controlled by diazepam, phenytoin, or phenobarbital. Castro Conte et al. [μg/kg per min, increasing by 0.5 to 1 μg/kg every 2 min, until a maximum of 18 μg/kg per min) controlled the seizures in 13 neonates refractory to the treatment with phenobarbital and phenytoin.A useful action of midazolam is the management of neonatal seizures refractory to conventional treatments. Hu et al.  reportede et al.  found thTroponin T is a prognostic indicator of postoperative recovery. Midazolam, propofol, or sevoflurane had similar efficacy in the production of troponin T in infants who underwent cardiac surgery .The information on the effects of midazolam and on the fate of this drug in neonates has been published in different journals during the last thirty years and it is scattered. The aims of this article are (1) to gather together and (2) to review the published data on (a) the metabolism and (b) the pharmacokinetics of midazolam in neonates, (c) the therapeutic effects, and finally (d) the adverse effects of midazolam in neonates. The main objective of this work is (e) to provide neonatologists with a tool that embraces all aspects of the clinical pharmacology of midazolam in neonates. Little is known about the effects and the fate of the drugs administered to neonates. This review was written to help the neonatologists, hoping that they will find some useful information and thus some advantages for their work, from reading the present paper.The bibliographic search was performed electronically using PubMed and EMBASE databases as search engines; September 2013 was the cutoff point. The following key words: “midazolam neonate,” “midazolam mechanism of action,” “midazolam therapy neonate,” “midazolam pharmacokinetics neonate,” “midazolam metabolism neonate,” “CYP3A4 midazolam neonate,” “CYP3A5 midazolam neonate,” “oral administration midazolam neonate,” “intranasal administration of midazolam neonates,” “midazolam seizures,” “midazolam anaesthesia neonate,” and “midazolam adverse effects neonate,” were used. The bibliography of each article was read carefully, and the selected articles were examined. In addition, the books NEOFAX: a Manual Used in the Neonatal Care, by Young and Mangum [μg/kg of midazolam over at least 5 min. Midazolam may also be administered intramuscularly. Repeat as required, usually Q2 to 4 hours. Dosage requirements are decreased by concurrent use of narcotics. For continuous intravenous infusion, give a 10 to 60 μg/kg midazolam per hour. Dosage of this drug may need to be increased after several days of therapy because of development of tolerance and/or increase of Cl. For Intranasal administration, give a 200 to 300 μg/kg midazolam per dose using 5 mg/mL injectable form. For sublingual administration, give a 0.2 mg/kg per dose of midazolam using 5 mg/mL injectable form mixed with small amounts of flavoured syrup. For Oral administration, give 250 μg per dose. For Anticonvulsant therapy; give intravenously a loading dose of 150 μg/kg over at least 5 min followed by a maintenance infusion dose of 60 to 400 μg/kg per hour (1 to 7 μg/kg per min).For sedation, Young and Neofax  suggest Development has a profound effect on the expression of phase I enzymes such as the cytochromes P450 (CYPs). Distinct patterns of isoform specific development CYP expression have been observed postnatally. Although the CYP content in the fetal liver equals about 30 to 60% of adult values , isoformIn adults, midazolam is metabolized rapidly ; the recPlasma Cl of midazolam is widely used as an in vivo surrogate measurement of CYP3A4 and CYP3A5 activities , 45. Cl Ince et al.  developeμg/kg) was administered intravenously (n = 15) or orally (n = 7). After intravenous administration, the median percentage of the dose excreted into the urine, over 6-hour intervals, was 0.44% (midazolam), 0.04% (1-hydroxymidazolam), and 1.57% (1-hydroxymidazolam glucuronide). After oral administration, the median percentage of the dose excreted into the urine was 0.11% (midazolam), 0.02% (1-hydroxymidazolam), and 1.69% (1-hydroxymidazolam glucuronide). The glucuronidation of 1-hydroxymidazolam is immature in preterm infants less than 2 weeks of postnatal age.The ontogeny of midazolam glucuronidation was studied in 22 preterm infants . MidazolThe Cl of midazolam was measured in 24 preterm infants and the mean was 1.8 (mL/kg per min) . In chilThe published literature on the pharmacokinetics of midazolam in neonates is scarce and the available information is heterogeneous because the work was done in healthy neonates , in critThe information on the pharmacokinetics of midazolam summarised in this review is organised on the neonate health status, and it is reported in (a) healthy neonates, (b) in critically ill neonates (c), and in infants undergoing extracorporeal membrane oxygenation (ECMO) and finally (d) the population pharmacokinetics of midazolam are reviewed in neonates. The pharmacokinetic parameters of midazolam are summarised in μg/kg) were studied in 24 healthy preterm infants, with a gestational age between 26 and 34 weeks [t1/2 was 6.3 hours (range: 2.6 to 17.7 hours), Cl was 1.8 mL/kg per min (range 0.7 to 6.7 mL/kg per min), Vd was 1.1 L/kg (range: 0.4 to 4.2 L/kg), and AUC0–∞ was 971 ng·h/mL (range: 248 to 2353 ng·h/mL). In the text, these values are referred to as the “normal values” or the “normal estimates.”The pharmacokinetics of intravenous midazolam  and Tmax was 6 hours (range: 1 to 12 hours). The median 1-hydroxymidazolam AUCt0– to midazolam AUCt0– ratio was low (0.09) and showed a large interindividual variation (range: <0.001 to 1). There were no relationships between 1-hydroxymidazolam pharmacokinetic parameters and midazolam Cmax, AUC, and t1/2. No 4-hydroxymidazolam could be detected.No significant relationships were detected between age  and midazolam Cl, Vd, and t1/2 was 73.6 weeks. Predicted Cl changes with age, based on this model, were in close agreement with physiologically based pharmacokinetic models. Previously published pharmacokinetic parameters can be used to develop maturation models that address gaps in current knowledge regarding the influence of age on a drug's disposition. If midazolam sedation target concentration of 100 ng/mL, similar to that given to adults, is assumed, we might anticipate steady-state infusion rates of 14 μg/kg per hour in neonates, 50 μg/kg per hour in a 1-year-old infant, 60 μg/kg per hour in a 5-year-old infant, and 50 μg/kg per hour in a 12-year-old child. Age-related pharmacodynamic differences that will affect dose and the impact of active metabolites on response have not yet been quantified.Anderson and Larsson  describet1/2 was 9.8 hours, after a 200 μg/kg bolus of midazolam, and 12 hours, after a constant infusion of 60 μg/kg per hour of midazolam [t1/2 is 6.3 hours. These estimates are 155% and 190%, respectively, of the normal values [Disease reduces the midazolam elimination rate in neonates. In critically ill neonates, undergoing mechanical ventilation, idazolam . In norml values .μmol/L, received an intravenous bolus of 100 μg/kg midazolam. t1/2, Cl, and Vd of midazolam of this girl were 6.3-fold longer, 2.6-fold greater, and 14.5-fold larger, respectively, [A 15.1-year-old girl, suffering from renal failure, with malignant hypertension and with a creatinine concentration of 691 ctively,  than thectively, . The patctively, .t1/2 and a Cl of midazolam of 3.8-fold longer and 1.8-fold greater, respectively, [A 1.1-year-old boy, suffering from pneumonia, received the CYP3A4 inhibitor erythromycin and the CYP3A4 substrate fentanyl. He had a ctively,  than thectively, .μg/kg per hour (range: 50 to 270 μg/kg per hour). The pharmacokinetic parameters of midazolam are summarised in P = 0.035). A significant negative correlation was found between disease severity and midazolam Cl corrected for body weight . These results suggest that increased disease severity is associated with reduced midazolam Cl. Likely, these results reflect a reduction of CYP3A activity.Vet et al.  determinAlternative explanations could be the altered protein binding, midazolam being highly bound to plasma proteins , or, leμg/kg per hour; the infusion lasted for 60.0 ± 23.3 hours. In these patients, t1/2 estimate was double the normal value [μg/mL; in two cases, the patients received fentanyl before midazolam. The metabolic ratio of midazolam to 1-hydroxymidazolam, was 16.1 ± 21.2 (n = 11) at 48 hours after the administration. Various drugs were coadministered with midazolam during the study; they were fentanyl (n = 7), antibiotics, in most cases betalactams and aminoglycosides (n = 13), albumin (n = 5), and vasopressin drugs (n = 3). Jacqz-Aigrain et al. [t1/2.The pharmacokinetics of midazolam was studied in 15 preterm infants undergoing mechanical ventilation for respiratory distress syndrome . Midazolal value . Hypotenal value . Mean syn et al.  concluden et al. , can inhμg/kg of midazolam [t1/2, Cl, and Vd were 6.5 ± 2.3 hours, 2.0 ± 1.2 mL/kg per min, and 0.9 ± 0.3 L/kg, respectively. The kinetic parameters in these infants were similar to those of normal values [Ten critically ill preterm infants, with a postnatal age of 2 to 5 days, received a single intravenous bolus dose of 200 idazolam . Midazoll values .μg/kg followed by an intravenous infusion of either 50 μg/kg per hour, for postoperative monitoring, or 100 μg/kg per hour for conscious sedation). In children, a large variability in the pharmacokinetic parameters of midazolam was observed. Midazolam is a substrate for CYP3A4 and CYP3A5 and the expression of these enzymes considerably varies in neonates [Ince et al.  performeneonates , 10, 11.The wide variability of midazolam pharmacokinetic parameters cannot be explained by age-related changes alone. Ince et al.  studied A new dosing schedule was designed, aiming for midazolam concentrations between 45 and 64 ng/mL for postoperative monitoring after procedural sedation  and betwExtracorporeal membrane oxygenation (ECMO) is a form of prolonged cardiopulmonary bypass used to support patients with life-threatening respiratory or cardiac failure . In neonECMO yields variability and lack of predictability in drug behaviour. The most common mechanisms by which ECMO affects pharmacokinetics are sequestration of the drug in the circuit and the increase of drug Cl and Vd , 56. TheAdequate sedation of neonates receiving ECMO is essential to allay the physical, emotional, and psychologic distress experienced during intensive care. Midazolam, a short-acting benzodiazepine derivative with rapid onset of action, is the sedative of choice and is preferred over other benzodiazepines because of its water solubility and the perceived rapid elimination .n = 10) received midazolam extracorporeally (into the circuit), via a pigtail catheter, whereas group 2 (n = 10) received the drug via a central or peripheral venous catheter. Midazolam was administered as a continuous infusion at a rate between 50 and 250 μg/kg per hour, initiated as soon as cannulation was achieved and extracorporeal blood flow was established. The pharmacokinetic parameters obtained by Mulla et al. [Twenty neonates with gestational age of 39.5 ± 1.9 weeks, postnatal age of 3.8 days, and body weight of 3,400 ± 600 g were enrolled in the study . The infa et al.  are summThe mean metabolic ratio was of 1-hydroxymidazolam to midazolam 0.17 (range: 0.03 to 0.9) and appeared to be significantly higher than that previously reported in non-ECMO infants (0.09), and it was similar to those observed in older children and adults .DeBerry et al.  determinμg/kg) was administered by a venoarterial catheter as a bolus infusion before cannulation. The median ECMO duration was 124 hours (range: 70 to 275 hours). When discomfort occurred, midazolam was started as a continuous infusion of 100 μg/kg per hour. The midazolam dose was incrementally adjusted (with steps of 100 μg/kg per hour) on the basis of the required level of sedation, which was objectified with the validated COMFORT behaviour scale. Once the patient was fully sedated, the midazolam infusion was interrupted until COMFORT scores indicated that additional sedation was necessary.Ahsman et al.  studied Midazolam Cl and Vd rapidly increased in 5 days of ECMO therapy. The kinetic parameters of midazolam are summarised in μg/kg per hour for the first 6 hours, after which, the infusion rate should be reduced to 150 μg/kg per hour. After 5 days, the infusion rate should be increased to 200 μg/kg per hour to compensate for the continual increasing of midazolam Cl and 1-hydroxymidazolam Cl. 1-Hydroxymidazolam glucuronide accumulated during ECMO, providing an increased proportion of the overall effect, up to 34% after 7 days of ECMO therapy. Large unexplained interindividual variability warrants careful titration of sedation effects. Ahsman et al. [To quickly reach stable serum concentrations of 400 ng/mL, the optimal dose regimen would be a continuous infusion of 300 n et al.  did not n et al. , 61.μg/kg per hour (range: 11 to 603 μg/kg per hour) to 109 infants; the infusion lasted for 6 to 171 hours (62 ± 31 hours). A bolus dose of 210 ± 239 μg/kg  was administered to 22 infants, and a combination of both was administered to 56 neonates. The mean midazolam doses required for critically ill neonates are lower than those required for wealthy neonates. Mean t1/2 was 9.9 hours, Cl was 1.2 ± 0.2 mL/min/kg, and Vd was 1.0 ± 0.2 L/kg [r = 0.89; P = 0.0001). The 531 measured plasma concentrations of midazolam ranged from 0 to 7,100 ng/mL; 105 samples were above 1,000 ng/mL. Midazolam was not detected in 19 samples. Aminoglycosides, aminopenicillins, and third generation cephalosporins increased the plasma midazolam concentrations for 73.8%, 72.2%, and 48.7%, respectively. Aminoglycoside treatment was associated with a 50% decrease in midazolam Cl.In literature there are three studies of midazolam population pharmacokinetics –34. One μg/kg was administered every 4 to 6 hours. Average parameter values for infants with birthweight of 1,000 g were as follows. Systemic Cl was 0.783 mL/min (CV = 83%) and Vd of central comportment was 473 mL (CV = 70%). t1/2 was not given. For infants with body weight more than 1,000 g, Cl was 1.24 mL/min (CV = 78%) and Vd was 823 mL (CV = 43%). The average population Cl in boys (0.642 mL/min) was less than in girls (0.808 mL/min). The level of significance of this difference was not given. The population mean Cl of overall patients was 0.938 mL/kg per min. The average Vd of overall infants was 1.15 L/kg, which agreed closely with the Vd of 1.0 L/kg in term and preterm infants [Lee et al.  studied  infants .μg/mL followed by a continuous infusion ranging from 50 to 400 μg/kg per hour, for 3.8 hours to 25 days for conscious sedation. t1/2, Cl, and Vd of midazolam were 5.5 ± 3.5 hours, 5.0 ± 3.9 mL/kg per min, and 1.7 ± 1.1 L/kg, respectively. Half of the patients had an age less than 6 months [The population kinetics of midazolam was studied in eighteen pediatric patients, aged from 2 days to 17 years . Midazol6 months . Mean Cl6 months , 62).The Cl reported by de Wildt et al.  is compa1-Hydroxymidazolam to midazolam ratio  is in aVery little is known about the pharmacodynamics of midazolam in infants and only two articles are available in literature , 63. Swaμg/kg per hour, for a period of 3.8 hours to 25 days [μg/kg per hour) were in agreement with other data from studies in pediatric intensive care patients [Twenty-one infants, aged from 2 days to 17 years, received a continuous infusion of midazolam from 50 to 400  25 days . A possi 25 days  recommen 25 days . As reflpatients , 71.Neonates can perceive pain when mechanically ventilated: this fact has been demonstrated by the physiological, biochemical, and behavioural changes indicative of pain and stress in newborn infants during mechanical ventilation . Pain inD. A. Rosen and K. R. Rosen  wrote a The American Academy of Pediatrics Committee on Drugs has issued guidelines categorizing pharmacological intervention into three levels: conscious sedation, deep sedation, and general anaesthesia . ConscioIntravenous infusion of benzodiazepines is important to assist ventilation and other potentially distressing procedures. Combined with intermittent or continuous infusion with opioids, the benzodiazepines provide smooth control of anxiety, pain, and agitation . The majμg/kg followed by a maintenance dose ranging from 37.5 to 100 μg/kg per hour over 47 hours. The sedation procedure was classified as a success if all the following clinical criteria were met: no agitation, no grimacing, and no crying facial expression before as well as during tracheal suctioning. The estimated success probability, updated after each patient, was <95% for all dose levels and led to allocation of the maximum loading dose (200 μg/kg). Based on the 23 patients studied, the final estimated probability of success was 76.9%  for the 200 μg/kg loading dose. The posterior success probability for the six doses 75, 100, 125, 150, 175, and 200 μg/kg corresponded to 27.7%, 32.7%, 38.1%, 47.3%, 52.5%, and 76.9%, respectively. Failures were recorded in 5 (25%) of 20 patients receiving a 200 μg/kg midazolam loading dose. No significant effect of sedation on pulse oximetry, oxygenation index, or triggering of ventilator breathing was evident after the onset of midazolam administration. No significant adverse effects were observed. Pneumothorax was reported in two children who had received a 200 μg/kg initial loading dose and were classified as “success” for sedation. A decrease of more than 30% for systolic, diastolic, and mean blood pressure was observed during the study inclusion in none, two (8.7%), and one (4.3%) children, respectively .Treluyer et al.  determinn = 24) or placebo (n = 22) as a continuous infusion [μg/kg per hour of midazolam. Infants below 33-week gestation received the same continuous infusion of 60 μg/kg per hour during the first 24 hours of treatment, followed by a continuous infusion of 30 μg/kg per hour of midazolam. The assigned treatment lasted for 5 days, but sedation with midazolam could be continued if prescribed by the attending neurologist. Midazolam gave a significantly better sedative effect than placebo (P < 0.05). Heart rate and blood pressure were reduced by treatment but remained within the normal range for gestational age and there was no effect on ventilatory indices. Hemodynamic instability requiring albumin and/or vasoactive drugs occurred in 14 infants, 8 of whom were in the midazolam group and 6 infants were in the placebo group. Intraventricular haemorrhage occurred in 8 infants in the midazolam group and 6 infants in the placebo group. Blood samples were obtained from 23 of 24 infants. Mean midazolam concentrations were 634 ± 234 ng/mL, 628 ± 327 ng/mL, and 543 ± 327 ng/mL at 24 and 48 hours and at the end of treatment, respectively. Substantial variability in plasma concentrations of midazolam was seen, but none of the infants had values below 200 ng/mL and only 2 infants  had a plasma concentration of midazolam above 1000 ng/mL. There was no correlation between sedation score and corresponding midazolam concentration. No midazolam-related adverse effects were noted. Continuous infusion of midazolam at doses adapted to gestational age induces effective sedation in newborn infants on mechanical ventilation, with positive effects on hemodynamic variables. Midazolam was given over only a few days and the limited effects on heart rate and blood pressure that were reported should not encourage long-term administration.Forty-six infants undergoing mechanical ventilation for respiratory distress syndrome were randomly assigned to receive midazolam  for analgesia and sedation [Midazolam was administered as a continuous infusion at a dosage of 50 to 400 sedation . The meaμg/kg followed by continuous infusion of 60 μg/kg per hour. Sixteen infants received placebo. The midazolam group had significantly better sedation than the placebo group from 18 to 24 hours after enrollment. At 48 hours, there were no significant differences in sedation between midazolam and placebo groups. None of the infants had hypotension on loading midazolam. Sedation provided by continuous infusion of midazolam appeared to be comparable to morphine alone in newborn infants on mechanical ventilation. These authors observed that the behavioural assessment was used to demonstrate the sedative effect of midazolam in ventilated newborn infants. Opioid and benzodiazepines combination is most commonly used to sedate newborns, but their use is limited by prolonged variable elimination of these drugs, leading to prolonged sedation. Midazolam produces a rapid and consistent effect because of its short t1/2. Hypotension has been reported as an adverse effect of midazolam in few infants after intravenous administration of midazolam [Arya and Ramji  performeidazolam , 78. Aryidazolam  did not When minimal sedation is required, intranasal midazolam is an excellent sedative . IntranaIntranasal midazolam use has been reported since 1988 . The intμg/kg (range: 300 to 800 μg/kg). Laceration repair was the most common procedure necessitating sedation (89%). Eleven patients (5.4%) required an additional sedative dose to complete the procedure. Ten of 11 patients received ketamine as the adjunctive sedative, and 1 patient required additional intranasal midazolam. There was 1 adverse event (0.5%). This was a minor desaturation episode following ketamine administration requiring a brief blow by oxygen. There were no adverse events in patients who received intranasal midazolam. These authors conclude that atomized intranasal midazolam is effective in providing anxiolysis to children undergoing minor procedures in the pediatric Department.Intranasal midazolam was studied by Lane and Schunk  using a μg/kg. Intranasal midazolam has a rapid onset and is more convenient than intravenous agents, because of its short duration. A dose of 200 μg/kg is safe and effective. In 142 of 162 cases (88%), midazolam demonstrated a positive effect; in 20 cases (12%), it was ineffective. The adverse effects were brief coughing, sneezing, or crying often occurring immediately after midazolam administration, but these were not considered adverse reactions unless they persisted. In 91 of 162 cases (56%), midazolam was used in conjunction with another agent. Fifteen adverse reactions (9%) occurred, including delay in awakening in 4 patients, reactive airway in 2 cases, desaturation and apnea in 4 cases, seizure in 1 case, agitation in 2 cases, and vomiting in 2 cases. Midazolam was the sole sedative in 71 of 162 (44%) cases. The use of midazolam to minimize the difficulty of starting an intravenous infusion constitutes the most common application. In 89% of the patients, it accomplished the desired effect. Of particular interest was the use of midazolam as a primary means of sedation since its intranasal route obviates the need for intravenous medication. An intranasally administered sedative with quick onset and short duration seems very innocuous.Harcke et al.  reviewedμg/kg was administered to 15 preterm infants with gestational and postnatal ages ranging from 26 to 31 weeks and from 3 to 13 days, respectively. Mean ± SD of body weight was 1076 ± 240 g. In 8 out of these 15 patients, the pharmacokinetics of intravenous midazolam was also studied. Apparent oral Cl, apparent Vd, and plasma t1/2 are given as median and range. Cl was 2.7 mL/kg/min (range: 0.64 to 15.5 mL/kg/min), Vd was 1.4 L/kg (range: 0.3 to 12.1 L/kg), and t1/2 was 7.6 hours (range: 1.2 to 15.1) hours. The absolute bioavailability was 0.49 (range: 0.12 to 1.0).Data describing midazolam disposition following oral administration to preterm infants are few. In literature, there are only two articles on the fate of midazolam following oral administration to neonates , 6. A siμg/kg midazolam. Pharmacodynamic measurements consisted of a COMFORT score at baseline and at 0.5, 1, 2, 4 and 6 hours after dose. A total of 24 infants were enrolled, of whom seven received both intravenous and oral midazolam, 13 infants received only intravenous midazolam, and 4 received only oral midazolam.In another study, De Wildt et al.  evaluateP = 0.003). No relationship was found between overall COMFORT scores or change in COMFORT score from baseline and midazolam. Diastolic blood pressure decreased significantly (approximately 11%) after intravenous, but not after oral, midazolam administration. No serious adverse events were reported. Midazolam administered as an intravenous infusion or oral bolus dose appears to be effective and well tolerated in a small majority of preterm infants. However, a considerable number of neonates did not respond to midazolam. The lack of response may be due to the fact that patients truly experienced therapeutic failure and/or consequent to the inability of the COMFORT score to adequately reflect sedation uniformly in sick preterm infants.Overall, mean COMFORT score decreased  significantly within 30 min after intravenous administration and within 1 hour after oral administration . If seizures continue, consider phenobarbital drip or lidocaine drip if not yet tried (unless phenytoin/fosphenytoin has been used).Slaughter et al.  reviewedThe neonatal period is one of the highest risk periods for seizures , which oμg/kg per hour for a period of 43 to 54 hours. Midazolam may be considered a safe and effective antiepileptic drug in refractory neonatal seizures of diverse aetiologies.Van Roij et al.  reviewedStatus epilepticus, a serious, life-threatening emergency, characterized by prolonged seizure activity, occurs most commonly in pediatric patients. Diazepam, phenytoin, or phenobarbital terminates seizures within 30 to 60 min, but there are patients who are refractory to standard therapy. Midazolam has emerged as a new treatment option. The review by Holmes and Riviello  comparedμg/kg per min, increasing by 0.5 to 1 μg/kg per min every 2 min until a favourable response or a maximum of 18 μg/kg per min). If the status epilepticus persisted after the initial bolus, another bolus of 100 to 150 μg/kg was administered 15 to 30 min later. Nonresponders to phenobarbital/phenytoin were controlled by midazolam.Castro Conde et al.  stated tLevel 1: rectal midazolam of 500 μg/kg, or 100 μg/kg, intravenously was administered. Level 2: after 10 min, 20 mg/kg phenytoin was administered intravenously. Level 3: after phenytoin load, 200 μg/kg midazolam was administered intravenously as a bolus, followed by a continuous infusion of 100 μg/kg per hour of midazolam, increased by 100 μg/kg per hour every 10 min, after extra loading of 100 μg/kg, to a maximum of 1 mg/kg per hour. Level 4: phenobarbital 20 mg/kg intravenously, or pentobarbital 2 to 5 mg/kg intravenous load, 1 to 2 mg/kg per hour of phenobarbital was continuously intravenously infused. Eighty-two of 122 infants (68%) received initial rectal diazepam. Cessation of epileptic activity was achieved with midazolam in 58 patients (48%). Midazolam is an effective agent for seizure control [The efficacy of a combination of midazolam and phenytoin in treating generalized convulsive status epilepticus was studied in 122 children with the median age of 24.4 months (range: 0.5 to 197 months) . Forty-t control . Several control , 102–104μg/kg per min, with an average of 4 μg/kg per min. There were no significant changes in serum sodium, potassium, calcium, or glucose in any of the patients. Four patients had recurrent seizures, which stopped after midazolam was reinstituted. Adverse effects included hypotension in 12 patients (38%), that was successfully controlled with dopamine and/or dobutamine, and transient urinary retention in 38% of infants. All the patients enrolled in the study had a successful management of seizures. These results suggest that midazolam is safe and effective for the treatment of uncontrollable seizures.Hu et al.  determinMidazolam is a highly effective antiepileptic agent, even in patients who have not responded to other benzodiazepines . Compareμg/kg and then maintained between 100 and 400 μg/kg per hour. After the loading dose, clinical seizures were no longer observed in four infants. The remaining became seizure free within one hour after the administration of midazolam. These results show that midazolam administrated by continuous intravenous infusion may be a valuable adjunct in the management of refractory neonatal seizures.Sheth et al.  studied McIntyre et al.  comparedMahmoudian and Zadeh  determinMany agents can depress the function of the central nervous system producing calming or drowsiness (sedation). Sedation may be performed with opioids , with the sedative-hypnotic midazolam and with the aesthetic propofol in ventilated neonates.Endotracheal intubation of premature infants is performed frequently in the “neonatal intensive care units” and delivery room. The procedure is associated with physiological and biochemical responses, and now there is strong evidence that premedication  improves the physiological stability, decreases the difficulty of procedure, and also reduces the potential for airway injury .μg/kg and 1 μg/kg remifentanil. The remaining 10 patients, with a gestational age of 31.5 ± 1.5 weeks and a body weight of 1523 ± 35 g, were treated with 2,000 μg/kg propofol and 1 μg/kg remifentanil. All neonates were in nasal continuous airway pressure therapy before intubation, with similar ventilatory parameters and degree of respiratory system. No statistical differences were observed as well between the drugs regarding pain and stress level before and after the intubation using the NIPS scale. No differences were observed in heart rate and blood pressure between the two drugs. Adverse effects such as hypotension were observed in two infants in each drug, and bradycardia was seen in one infant in the propofol group. The present findings did not show any difference in the quality of intubation, presence of adverse effects, changes on the hemodynamic variables, and quality of sedation and analgesia achieved when midazolam or propofol was used as a hypnotic associated with remifentanil as premedication for tracheal intubation in preterm neonates. The pharmacokinetics of propofol was studied in 25 neonates after a bolus injection of 3000 μg/kg [Penido et al.  compared00 μg/kg  and founμg/kg) and midazolam (500 μg/kg) 30 min before induction of anaesthesia. Anaesthesia was induced with sevoflurane (1 μg/kg), followed by a bolus of sufentanil (1 μg/kg) and pancuronium (200 μg/kg). The groups were comparable with respect to sex, age, weight, type of surgery, cardiopulmonary bypass, aortic cross clamp, and circulatory arrest time. The values for acyanotic patients were similar with midazolam, propofol, and sevoflurane. The corresponding values for cyanotic patients were similar to the three anaesthetics. There was a significant correlation between ventilator hours and troponin T concentrations, at the time of 8 hours, in the sevoflurane group (r = 0.45) and midazolam group (r = 0.50) but not in the propofol group. The present findings show that the postoperative production of troponin T, in pediatric patients undergoing cardiac surgery, is similar to midazolam, propofol, or sevoflurane anaesthesia.Malagon et al.  comparedμg/kg per hour. Fifteen of the 24 children received a combination of midazolam with fentanyl; the latter was infused at a rate of 0.5 to 2 μg/kg per hour. The mean duration of fentanyl infusion was 64 hours (range: 20 to 188 hours). No cardiovascular or respiratory adverse effects were seen. After stopping the fentanyl infusion, the children were sedated with midazolam. With midazolam as the only sedative drug, a significantly (P < 0.05) higher mean infusion rate of midazolam was necessary to reach a reliable sedation and it was of 209 μg/kg per hour (range: 100 to 500 μg/kg per hour). Mean serum concentration of midazolam decreased from 513 μg/L  to 330 μg/L  after 4 to 5 days. Midazolam Cl increased in relation to the duration of midazolam therapy . In the early phase of midazolam therapy, Cl of midazolam was 5.8 + 3.8 mL/kg per min and increased significantly by 133% to 13.6 + 10.6 mL/min per min in the late phase of therapy. The reason for the increase of midazolam Cl may be either an increase of the Vd or an increase of the hepatic Cl due to an enzyme induction of midazolam or by increasing hepatic blood flow during a treatment over a longer period. During the first 24 to 72 hours of midazolam therapy, the sedation score was significantly correlated with the serum midazolam concentration . The desirable sedation score could be achieved during this time period at a serum concentration between 100 and 500 ng/mL with an infusion rate of 100 to 400 μg/kg per hour. Midazolam seems to guarantee reliable sedation and anxiolytic action. The evaluation of the sedation score showed that reliable sedation can be achieved at a midazolam infusion of 100 to 400 μg/kg per hour and at a serum midazolam concentration between 100 and 500 ng/mL. In combination with fentanyl, the midazolam infusion rate could be held at a lower level. Continuous intravenous infusion of fentanyl and midazolam has been shown to provide satisfactory analgesia.Hartwig et al.  studied n = 79; 1.05 ± 0.88 mg/kg per hour), midazolam , or both . In 18.5% of patients there were complex cardiac lesions. Pre- and postprocedure systolic and diastolic mean blood pressure were 72 ± 14 and 68 ± 12 mmHg, respectively. Mean procedure time was 79 ± 36.2 min. The anaesthesiologist's assistance was required by the cardiologist in 21 procedures. Pre- and postprocedure O2 saturation were 93.19 ± 8.72 and 93.63 ± 8.3, respectively. The mortality was zero. The two groups were not different in relation to the drug use (P = 0.283) or the complexity of the cardiac lesion (P = 0.051). However, there was significant difference between the two regarding the need for supporting drugs  or oxygen treatment . These authors conclude that low dose of ketamine and midazolam can be administered safely to most pediatric patients by the cardiologist, who can safely predict the need for an anaesthesiologist.Ketamine and midazolam are commonly used in children undergoing cardiac catheterization. Jobeir et al.  reviewedMidazolam is incompatible with fat emulsion, albumin, ampicillin, bumetanide, cefepime, ceftazidime, dexamethasone, fosphenytoin, furosemide, hydrocortisone succinate, micafungin, nafcillin, dimenhydrinate, perfenazine, ranitidine hydrochloride, thiopental, prochlorperazine edisylate, and sodium bicarbonate , 111. KeThere have been reports of life-threatening adverse respiratory and cardiovascular events occurring after administration of midazolam. Pain, tenderness, and thrombophlebitis have occurred following injection of midazolam. Hiccups have been reported. Death due to respiratory depression, hypotension, or cardiac arrest has been reported in patients given intravenous midazolam for conscious sedation .A receptors in the neonate [Midazolam is not free from adverse effects when administered to neonates. The first intravenous loading dose of midazolam administered to premature infants not infrequently causes respiratory depression, with hypotension, a fall in cerebral blood flow, and paradoxical agitation . The par neonate .Respiratory depression and hypotension are common when midazolam is used in association with narcotics or following rapid bolus administration. Hypotension has also been reported after continuous infusion of midazolam. The percentage of infants who develop hypotension varies with the studies. Seizure-like myoclonus has been reported in 8% of premature infants receiving continuous infusion . This alμg/kg midazolam, but the most disturbing observation was the appearance of involuntary epileptiform movements lasting for 15 to 30 sec. The pathogenesis of this phenomenon is speculative, but perhaps in these preterm infants the decrease in arterial pressure and heart rate has also an impact on cerebral blood flow or there is a direct effect of midazolam on the brain.Van den Anker and Sauer  observedNg et al.  recentlyThe major adverse effects associated with continuous infusion of midazolam are related to tolerance and an associated abstinence or withdrawal syndrome , 114. Wiμg/kg midazolam. No change in heart rate occurred during the study period, while mean arterial blood pressure decreased by 3 mmHg, 5 min after midazolam administration, compared to baseline values. A nonspecific fall in transcutaneous PCO2 was seen at 20 min after midazolam administration. Mean cerebral blood flow decreased from baseline by 12% at 5 min after midazolam administration then returned to predose values. Midazolam concentrations in the therapeutic range were proved to be effective in sedation of pediatric intensive care infants. As only minor cerebral and hemodynamic effects were found with the use of midazolam in stable ventilated preterm infants, it appears that midazolam is a safe, short-term sedative agent.Harte et al.  evaluateμg/kg midazolam, followed by a constant infusion of 200 μg/kg per hour, to 11 ventilated premature infants, caused changes in cerebral oxygenation and hemodynamics [μg/kg midazolam, followed by a continuous infusion of 150 μg/kg per hour. Harte et al. [μg/kg of midazolam to 10 infants with a birthweight ≤1500 g.The administration of a loading dose of 200 dynamics . Van Leudynamics  reportede et al.  reportedt1/2 of midazolam is 22-fold shorter than that of diazepam [t1/2, Cl, and Vd are 1.9 hours, 6.6 mL/kg per min, and 1.1 L/kg, respectively. In healthy preterm infants, t1/2 is 3.3-fold longer, Cl is 4.4-fold smaller, and Vd is 1.1 L/kg in adult and neonates. The rate of midazolam elimination is slower in infants than in adults. Midazolam is mainly eliminated by metabolism in adults. It is hydroxylated in position 1 to form 1-hydroxymidazolam by CYP3A4 and CYP3A5. These enzymes surge in the liver during the first weeks of life [t1/2 and the slower Cl observed in neonates than in older infants and adults.The present paper reviews the clinical pharmacology of midazolam; most of the work was performed in neonates, and several data have been obtained in children. Benzodiazepines provide effective control of patient agitation without imposing a significant load on the cardiovascular system. Midazolam is a short-acting benzodiazepine with rapid onset of sedation and is preferable to diazepam. diazepam . In adul of life , 9, 11 aChildren, with an age greater than 2 years, had a Cl of midazolam that ranged from 5.8 to 13.6 mL/kg per min (mean ± SD = 9.6 ± 3.5 mL/kg/min) , 47. Theμmol/L and treated with midazolam, had a t1/2, Vd, and Cl of this drug 6.3-fold longer, 14.5-fold greater, and 2.6-fold larger, respectively, [Disease affects the pharmacokinetics of midazolam. A patient suffering from renal failure, with malignant hypertension, and having a creatinine concentration of 691 ctively,  than thectively, . Multiplctively, . The sevμg/kg per hour [t1/2 1.9-fold longer than the normal value [t1/2. The long t1/2 of midazolam observed by Jacqz-Aigrain et al. [t1/2, Cl, and Vd of midazolam [Fifteen preterm infants, requiring mechanical ventilation, were treated with midazolam continuous infusion of 60 per hour . They haal value . Some ofal value . Fentanyn et al.  may reflidazolam , 119.t1/2 was 1.6-fold greater [t1/2 in neonates. The infants studied by Lee et al. [t1/2 observed by Lee et al. [t1/2, Cl, and Vd were 5.5 ± 3.5 hours, 5.0 ± 3.9 mL/kg per min, and 1.7 ± 1.1 L/kg, respectively. These parameters were 0.87-fold lower, 3.0-fold higher, and 1.5-fold larger than the normal values. These data suggest that the Cl of midazolam increases with the neonatal development.In literature, there are three studies on the population pharmacokinetics of midazolam –34. Burt greater  than the greater  and 2.2- greater  than nor greater . These fe et al.  had a sme et al. . The lone et al.  might, aThe data on midazolam pharmacodynamics in neonates are few and do not allow any conclusion. De Wildt et al.  did not Neonates may undergo uncomfortable procedures and may experience moderate to severe pain in the “neonatal intensive care unit.” The availability of an appropriate sedative that reduces the stress and pain is desirable. Midazolam is an effective sedative and represents a convenient choice among the sedatives because of its fast onset of action and the rapid termination of effect. The use of midazolam for newborn sedation remains empirical, and further research on the effectiveness and safety of midazolam in ventilated neonates is needed before its employment can be considered in routine clinical use , 111.μg/kg (range: 300 to 800 μg/kg). The bioavailability of intranasal midazolam ranged from 50% to 83% [Sedation of neonates with midazolam may be performed by intravenous administration (bolus or continuous infusion) or by intranasal administration when minimal sedation is required . The med% to 83% . A secon% to 83% , 6. No iμg/kg midazolam, the final estimated probability of success is 76.9%. Using lower midazolam concentrations, the probability of success decreased.Treluyer et al.  determinμg/kg per hour. Infants, below 33 weeks of gestation, received the same continuous infusion rate during the first 24 hours of treatment, followed by a continuous infusion of 30 μg/kg per hour of midazolam. Mean midazolam concentrations were 634 ± 234 ng/mL, 628 ± 327 ng/mL, and 543 ± 327 ng/mL at 24 and 48 hours and at the end of treatment. Remarkable variability in midazolam plasma concentrations was observed, but none of the infants had levels of midazolam below 200 ng/mL and only 2 infants had a plasma concentration of midazolam above 1000 ng/mL.Mulla et al.  administμg/kg per hour produced midazolam concentrations sufficient for sedation of infants [μg/kg, followed by a continuous infusion of 60 μg/kg per hour, to 17 infants with a body weight <2000 g, undergoing mechanical ventilation. No significant adverse effects were observed. The course of mechanical ventilation was not influenced by the use of midazolam. These authors observed that the behavioural assessment was used to demonstrate the sedative effect of midazolam in ventilated infants.A dose of midazolam between 50 and 400  infants . Dosage  infants  administμg/kg was administered to infants (n = 15) with gestational age ranging from 26 to 31 weeks. The apparent oral Cl and Vd and the plasma t1/2 (given as median) were 2.7 mL/kg per min, 1.4 L/kg, and t1/2 7.6 hours, respectively. These values are not considerably different from the normal values. The absolute bioavailability “F” of midazolam was 0.49.Data describing midazolam pharmacokinetics following oral administration to neonates are few; only two articles are available in literature , 6. A siThe neonatal period is one of the highest risk periods for seizures , which oThe efficacy of a combination of midazolam and phenytoin, in treating generalized convulsive status epilepticus, was studied in 122 children with the median age of 24.4 months . Forty-tμg/kg followed by a continuous infusion of 1 to 15 μg/kg per min. Twelve patients (38%) developed hypotension. Midazolam emerged as a treatment option against the status epilepticus in patients who did not respond to diazepam, phenytoin, or phenobarbital [Midazolam successfully managed seizures in 32 neonates who did not respond to diazepam, phenytoin, or phenobarbital . Midazolbarbital . Comparebarbital .Many drugs depress the function of the central nervous system producing sedation in ventilated infants. They are the opioids , the sedative-hypnotic midazolam, and the anaesthetic propofol. Mahmoudian and Zadeh  did not μg/kg midazolam followed by an infusion of this drug at a rate of 100 μg/kg per hour. Fifteen of the 24 infants received a combination of midazolam with fentanyl. The latter was infused at a rate of 0.5 to 2 μg/kg per hour. No cardiovascular or respiratory adverse effects were seen. Midazolam guarantees reliable sedation and anxiolytic action. With midazolam, as the only sedative drug, a significantly higher mean infusion rate of 209 μg/kg per hour (range: 100 to 500 μg/kg per hour) was necessary to reach a reliable sedation. In the early phase of midazolam therapy, Cl was 5.8 ± 3.8 mL/kg per min and increased by 133% to 13.6 ± 10.6 mL/kg per min in the late phase of therapy.Hartwig et al.  experienμg/kg per hour (n = 79) and midazolam infusion was 140 ± 90 μg/kg per hour (n = 35). There were no differences between the two groups regarding the need for supporting drugs or oxygen treatment. In 18.5% of patients there were complex cardiac lesions. No difference was found in relation to the drug used (P = 0.283) or the complexity of cardiac lesions (P = 0.051). These authors conclude that low dose of ketamine and midazolam can be administered safely to most pediatric patients by the cardiologist, who can safely predict the need for an anaesthesiologist.Ketamine was infused at a rate of 1050 ± 880 A receptors present in the neonate [Life-threatening adverse respiratory and cardiovascular events have been reported in adults after midazolam administration. Pain, tenderness, and thrombophlebitis have occurred following intravenous midazolam administration . In neon neonate .μg/kg midazolam reduces the mean arterial blood pressure by 12% at 5 min after midazolam [μg/kg per min, to treat seizures. Twenty-seven percent of the infants receiving a continuous midazolam infusion of 60 μg/kg per hour developed hypotension [μg/kg per hour [The major adverse effects associated with continuous infusion of midazolam are related to tolerance and an associated abstinence or withdrawal syndrome , 76, 112idazolam . Hypotenidazolam  observedotension . These iper hour .t1/2 of midazolam. It is likely that these effects are due to the lowering of CYP3A4 and CYP3A5 activities. We feel that further research is required to ensure that the doses of midazolam recommended for the treatment of neonates are evidence-based.In conclusion, midazolam is a safe and effective drug, which may be used as a sedative or as an antiepileptic for the treatment of infants who are refractory to standard antiepileptic therapy. Midazolam is particularly useful in the treatment of the status epilepticus in infants. Midazolam is also used as an anaesthetic, but information about midazolam as an anaesthetic is lacking in neonates. Midazolam is extensively hydroxylated by two cytochrome P-450 forms, namely, CYP3A4 and CYP3A5. These enzymes surge in the liver during the first weeks of life. Neonates have a lower expression of these enzymes than older infants and adults. Consequently, the metabolic rate of midazolam is lower in neonates. Diseases affect the pharmacokinetics of midazolam; they may reduce the Cl or prolong


Presenilin-1 (PS1) is the active component of the amyloid precursor protein cleaving γ-secretase complex. PS1 protein is a transmembrane protein containing multiple hydrophobic regions which presence in cerebrospinal fluid (CSF) has not been measured to date. This study assesses whether PS1 and other components of the γ-secretase complex are present in CSF.post-mortem and lumbar ante-mortem CSF, and plasma as 100–150-kDa hetero-complexes containing both the N- and C-terminal fragments (NTF and CTF) of the protein. Immunoprecipitation and immunoblotting with different antibodies confirmed the identity of the PS1 species. The γ-secretase components, APH-1 (anterior pharynx-defective 1) and PEN-2 (presenilin enhancer 2), as well as presenilin-2 (PS2) fragments, co-exist within these CSF complexes, while nicastrin is not detected. These CSF-PS1 complexes differ from active γ-secretase membrane-complexes, and may represent nonspecific aggregation of the PS1 protein. Levels of PS1 complexes are increased in CSF samples from autopsy-confirmed Alzheimer’s disease (AD) cases and were found to be more stable than complexes in CSF from control subjects. Despite similar levels of total PS1 in CSF from probable AD patients and cognitively normal subjects, an increased proportion of highly stable PS1 complexes were observed in AD CSF.Here, we show that PS1 is present in ventricular Our data suggest that fragments of the PS1 protein present in CSF as complexes may be useful as a biomarker for AD. The major pathological hallmarks of Alzheimer’s disease (AD), the β-amyloid peptide (Aβ) and the abnormally hyperphosphorylated protein tau (P-tau), are also recognized biomarkers which have been extensively investigated (for a review see ). HoweveOther logical AD biomarker candidates are proteins involved in the pathological processing of the large transmembrane protein, the amyloid precursor protein (APP), leading to β-amyloid formation in the AD brain. The Aβ polypeptide is generated by processing of APP, through the successive action of two proteolytic enzymes, β-secretase and γ-secretase . The majTo our knowledge, the presence of presenilins in CSF has not been reported to date. In this study we investigated if PS1 is detectable in CSF and if altered levels of this protein reflect the pathological condition.post-mortem CSF samples by Western blotting using different anti-PS1 NTF and CTF antibodies  , with veDifference in assay conditions, such as the presence of detergents, can affect the molecular interactions necessary for PS1 complex formation and its subsequent detection . PS1 aggInterestingly, under the conditions used for PS1 complex detection, PEN-2 and APH-1 immunoreactivities in CSF displayed similar electrophoretic banding patterns. However the traces of nicastrin that were detected were not part of the high molecular mass presenilin complexes , and were resolved as a 29-kDa band by Western blotting using an NTF-PS1 antibody under denaturing conditions. Brain-PS1 complexes were also identified in denser fractions, close to the sedimentation rate of catalase (molecular mass ~232 kDa). Interestingly, these large PS1 complexes were resolved as ~50-kDa heterodimers. However, CSF-PS1 complexes which sediment close to alkaline phosphatase were resolved as stable 100-150 kDa complexes, instead of monomeric NTF-PS1 . New aliquots of the same CSF samples were immunoblotted with an anti–CTF-PS1 antibody . The minor form of PS1 (29-kDa) was decreased in AD compared to NDC subjects ; while the 20 kDa-band corresponding to monomeric PS1-CTF was only very weakly detected and not quantified.To assess whether PS1 levels are altered in AD, we analyzed ventricular Fractionation on sucrose density gradients followed by Western blotting under denaturing conditions revealed that the highly stable 100-150 kDa complexes were more abundant in AD samples, while less stable complexes that sediment in denser fractions were mostly detected in NDC samples. This difference enabled us to define a quotient between highly stable complexes  and unstable complexes , allowing us to fully discriminate between the two groups  and with P-tau levels . In the NDC group, age trends to positive correlates with the quotient of the PS1 complexes .We have demonstrated that high molecular mass PS1 complexes are increased in post-mortem AD CSF. These PS1 complexes were also assessed in lumbar CSF from probable AD and control subjects. Measurement of classical AD biomarkers displayed elevated CSF T-tau and P-tau and low levels of Aβ42 in probable AD samples Figure . As expe490 of human BACE1 protein). Using this antibody, we have evaluated BACE1 levels by Western blotting in the same set of post-mortem CSFs. Full-length BACE1 was significantly elevated in AD patients compared with controls , or for AD cases . No change was observed in full-length BACE1 levels evaluated in lumbar CSF from probable AD and control subjects , Fundación Hospital de Alcorcón  and Fundación AlzheimUr . Samples contaminated with blood were excluded from analysis. CSF was centrifuged at 1000 × g for 15 min to eliminate cells and insoluble material prior to biochemical analyses. AD cases  were selected based on clinical history of dementia and neuropathological CERAD diagnosis [Ventricular CSF was obtained iagnosis . SamplesMicrosomal membranes were solubilized from 0.2 g of human frontal cortex  in buffer containing 0.5% dodecylmaltoside, 20% glycerol, and 25 mM Bis-Tris/HCl, pH 7.0, as previously described . γ-SecreLumbar CSF samples were obtained from 12 patients  with mild-moderate AD, and 12 healthy volunteers  from Huddinge University Hospital . All AD patients fulfilled the NINCDS-ADRDA criteria for “probable” AD . ControlPlasma samples from five healthy volunteers (76 ± 1 years) were collected in heparinized tubes at the Hospital General Universitario de Elche and separated from whole blood by centrifugation at 3000 × g for 15 min at 4°C. CSF and plasma samples were aliquoted and frozen at -80°C until use.PS1 cKO) and five littermates in a C57BL6/129 hybrid background. In these cKO mice, PS1 expression has been selectively eliminated in glutamatergic neurons from the postnatal forebrain at P18, and at 2–3 months of age revealed no detectable brain PS1 [CSF and plasma samples were also collected from four 3 month-old PS1conditional knockout mice (rain PS1 . CSF (4-rain PS1 . Blood w® (Invitrogen) according to the manufacturer’s instructions.SH-SY5Y cells  were grown in 6 well plates and transfected with a construct encoding full-length PS1   prior to gel electrophoresis (25 μg of protein after protein depletion). The separated proteins were transferred to nitrocellulose membranes (Schleicher and Schuell Bioscience GmbH) and probed with PS1 antibodies to N-terminal amino acids 1-65 , amino acids 21-34 (from Thermo Scientific), amino acids 1-20 , or plasma samples (0.5 μL) were denatured at 50°C for 15 min or alternatively at 98°C for 5 min for other analyses. For BACE1 analysis CSF samples were denatured always at 98°C for 5 min. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under fully reducing conditions. For blue-native gel electrophoresis, samples were analyzed as previously described  and Natidy 98/1, ); amino dy 98/1, ). CSF prdy 98/1, . BACE1 aImmunoprecipitations were performed at 4°C by incubating 150 μL of CSF overnight with the primary N-terminal PS1 antibody 98/1, or alternatively with an antibody from Thermo Scientific, previously coupled to protein A-Sepharose by Dimethyl pimelimidate dihydrochloride (Sigma-Aldrich Co). Precipitated proteins were washed with PBS and eluted with 0.1 M glycine buffer at pH 2.5. After pH neutralization, supernatants were denatured in Laemmli sample buffer at 50°C for 15 min and subjected to SDS-PAGE, and separated proteins were transferred to nitrocellulose membranes. Blots were incubated with N-terminal  and C-terminal (00/2) PS1 antibodies.g in a continuous sucrose density gradient (5-20%) for 4 hr at 4°C in a Beckman TLS 55 rotor. CSF aliquots (50 μL) were carefully loaded onto the top of the gradient containing 2 mL of 0.15 M NaCl, 50 mM MgCl2 and 0.5% Brij 97, in 50 mM Tris-HCl (pH 7.4). After centrifugation ~14 fractions were gently collected from the top of the tubes. Enzyme markers of known sedimentation coefficient, β-galactosidase, catalase and alkaline phosphatase were used in the gradients to determine the approximate sedimentation coefficients.PS1 complexes were analysed by ultracentrifugation at 250,000 × γ-Secretase activity was assayed as reported elsewhere  using anTotal tau (T-tau), phosphorylated tau (P-tau) and Aβ1-42 (Aβ42) in CSF were determined using specific enzyme-linked immunosorbent assays (ELISA) .t test (two-tailed) or Mann-Whitney U test for single pair-wise comparisons and determination of exact p values. Results are presented as means ± SEM. Correlation between variables was assessed by linear regression analyses. p values < 0.05 were considered significant.All data were analyzed in SigmaStat  by Student’s This study was approved by the ethics committee of the Miguel Hernandez University and was carried out in accordance with the Declaration of Helsinki.Aβ: β-amyloid peptide; Aβ42: Aβ1-42; AD: Alzheimer’s disease; APH-1: anterior pharynx-defective 1; BACE1: beta-site APP cleaving enzyme 1; CTF: C-terminal fragment; CSF: Cerebrospinal fluid; NDC: Non-demented control; NTF: N-terminal fragment; PEN-2: Presenilin enhancer 2; PS1: Presenilin-1; PS2: Presenilin-2; P-tau: Hyperphosphorylated tau; SEM: Standard error of mean; T-tau: Total tau.All authors have contributed to the work and agree with the presented findings.MSGA and JSV submitted a patent application for the use of PS1 complexes as an AD biomarker, as described herein. Appropriate procedures were followed to obtain approval from local ethics committees.post mortem CSF samples. JA provided freshly collected ante-mortem CSF samples. CAS provided PS1 cKO mice material and tested antibodies. NA provided CSF samples from AD and control subjects and supplied clinical data. KB designed experiments. JSV designed experiments, analyzed data and wrote the manuscript. All the authors participated in the preparation of the manuscript. All the authors contributed to the discussion of results and participated in the preparation of the manuscript. All authors read and approved the final manuscript.MSGA designed experiments, generated and analyzed data . MLC generated and analyzed data . GB generated and analyzed data (IP and ELISA). AR provided brain and


Little is known of vitamin D concentration in cerebrospinal fluid (CSF) in Alzheimer´s disease (AD) and its relation with CSF acetylcholinesterase (AChE) activity, a marker of cholinergic function.A cross-sectional study of 52 consecutive patients under primary evaluation of cognitive impairment and 17 healthy controls. The patients had AD dementia or mild cognitive impairment (MCI) diagnosed with AD dementia upon follow-up (n = 28), other dementias (n = 12), and stable MCI . We determined serum and CSF concentrations of calcium, parathyroid hormone (PTH), 25-hydroxyvitamin D (25OHD), and CSF activities of AChE and butyrylcholinesterase (BuChE). P < 0.05), and CSF AChE activity was decreased both in patients with AD (P < 0.05) and other dementias (P < 0.01) compared to healthy controls. None of the measured variables differed between BuChE K-variant genotypes whereas the participants that were homozygous in terms of the apolipoprotein E (APOE) ε4 allele had decreased CSF AChE activity compared to subjects lacking the APOE ε4 allele (P = 0.01). In AD patients (n=28), CSF AChE activity correlated positively with CSF levels of total tau (T-tau)  and phosphorylated tau protein (P-tau) , but CSF activities of AChE or BuChE did not correlate with serum or CSF levels of 25OHD.CSF 25OHD level was reduced in AD patients (In this pilot study, both CSF 25OHD level and CSF AChE activity were reduced in AD patients. However, the lack of correlations between 25OHD levels and CSF activities of AChE or BuChE might suggest different mechanisms of action, which could have implications for treatment trials.  Calcium influx into cells is a mediator of cellular metabolism, but unbuffered intracellular calcium could be neurotoxic for brain cells . CirculaThe vitamin D receptor (VDR) is abundantly expressed in the human brain . In patiVDR polymorphisms or deficiency of vitamin D could be risk factors for cognitive decline as well as AD ,9. In syThere is a loss of central cholinergic neurons in severe AD . In earlCholinesterase inhibitors are, with moderate effect, used to treat AD. The extent to which treatment with vitamin D is of additional value in this patient group remains to be established. In this study, we determined calcium and vitamin D status in serum and CSF as well as the relations with CSF activities of AChE and BuChE in a well characterized mono-center cohort of patients with cognitive impairment and in matched healthy controls. We also studied whether there were associations with CSF levels of AD biomarkers. 2, and waist:hip ratio 0.65-0.90 in women and 0.70-0.95 in men. Exclusion criteria were serum creatinine > 175 mmol/L, diabetes mellitus, previous myocardial infarction, malignancy including brain tumor, subdural hematoma, ongoing alcohol abuse, medication with steroids, and previous or present medication with AChE inhibitors. The study participants as well as AD CSF biomarkers have been reported previously . The stuControl subjects without any subjective symptoms of cognitive dysfunction were recruited contemporaneously from the same geographical area among spouses of the included patients and by advertisements in local newspapers. Totally, 60 patients (30 men and 30 women) and 20 healthy controls (10 men and 10 women) were recruited. However, in this analysis, 11 subjects  receiving medication with calcium or vitamin D were excluded. Therefore, 52 patients (29 men and 23 women) and 17 controls (9 men and 8 women) were included in the present study.P - value = 0.09), there were non-significant tendencies that a higher number of patients with other dementias had been recruited during winter and that SMCI patients had been recruited mainly during winter and autumn.The patients and the control were recruited at various times of the year. AD patients : 6 (21%), 9 (32%), 8 (29%), 5 (18%); patients with other dementias: 5 (42%), 2 (17%), 3 (25%), 2 (17%); SMCI patients: 5 (42%), 2 (17%), 0 (0%), 5 (42%); healthy controls: 5 (29%), 9 (53%), 1 (6%), 2 (12%). Thus, although there were no statistically significant difference between the study groups , criteria. Patients with dementia were classified as suffering of AD , vascula1-42 (Aβ1-42), total-tau (T-tau) or phosphorylated tau protein (P-tau) (data not shown). During the follow-up visits, 12 MCI patients remained in stable cognitive function (SMCI). Others progressed, during the follow-up period, to dementia and were diagnosed with AD (n = 6) or VaD (n = 3). MCI patients diagnosed with AD on follow-up visits did not differ in CSF levels of Aβ1-42, T-tau and P-tau from patients with established AD at baseline (data not shown). Totally, the study population consisted of AD dementia or MCI diagnosed with AD dementia upon follow-up (n = 28), other dementias (n = 12), SMCI (n = 12), and healthy controls (n = 17) (Mild cognitive impairment (MCI) was diagnosed in patients with cognitive impairment that did not fulfill the criteria for dementia . Patient(n = 17) .The study was approved by the ethical committee of Göteborg University. Ethical approval number: S 496-99. The specialist physician (P.J.) gave written information of the study and explained the study protocol to the controls as well as to the patients and caretaker if available. All participants provided both oral and written informed consent. A next of kin, caretaker or guardian consented on behalf of patients if the capacity to consent was compromised. However, in all cases, the patient's own opinion was asked and considered, and the patient was recruited in the study only when he or she agreed with this. The ethical committee approved this informed consent procedure. The study was conducted according to the principles in the Declaration of Helsinki.Before the test day, a mini-mental state examination (MMSE)  was perfAll CSF samples were collected by lumbar puncture in the L3/L4 or L4/L5 interspace at the standardized time point 08:30-09:00 h. The first 12 mL of CSF was collected in a polypropylene tube and immediately transported to the local laboratory for centrifugation at 2.000g at +4°C for 10 minutes. The supernatant was pipetted off, gently mixed to avoid possible gradient effects, and aliquoted in polypropylene tubes that were stored at -80 °C pending biochemical analyses, without being thawed and re-frozen. CSF and blood samples were obtained at the same visit. Blood samples were drawn in the morning in the fasted state and serum was prepared by centrifugation after coagulation at room temperature for 15-30 min, aliquoted and stored in cryotubes at -80 °C pending biochemical analyses, without being thawed and re-frozen.All biochemical analyses were performed with the analyst blinded to the clinical diagnoses and other clinical information. All analyses were performed at one occasion. Total calcium concentrations in serum and CSF were measured using the o-cresolphthalein method on a Roche Cobas c501 instrument . Serum and CSF levels of total 25OHD were analyzed using the Vitamin D Total immunoassay on a Roche Cobas e601 instrument (Roche Diagnostics). Serum levels of PTH were measured using an intact PTH immunoassay of sandwich type on an ARCHITECT i2000 instrument . CSF concentrations of PTH were below the lower detection limit of the assay (3 ng/L). CSF AChE and BuChE activities were determined using in house assays as previously described in detail . We then performed gender-specific analyses using the Kruskal-Wallis test to compare the four study groups with respect to serum 25OHD level. In men analyzed separately, there was no between-group difference (P = 0.19) whereas in women, serum 25OHD level differed between the study groups (P = 0.01). Post hoc analyses using the Mann-Whitney U test showed lower serum 25OHD level in female AD patients [36.0 (27.0-44.8) nmol/L] than in female SMCI patients  and female controls , but otherwise there were no difference between groups in women.In the total study population (n=69), we investigated whether levels of the measured variables differed between men and women. Only serum 25OHD level differed between genders with lower values in women [median (25th -75th percentile): 40.5 (34.3-53.5) nmol/L] compared to men [52.0 (41.0-56.3) nmol/L, In the present study, CSF 25OHD level was reduced in AD patients, and CSF AChE activity was decreased both in patients with AD and other dementias compared to healthy controls. None of the measured variables differed between BuChE K-variant genotypes whereas the participants that were homozygous in terms of the APOE ε4 allele had decreased CSF AChE activity compared to subjects lacking the APOE ε4 allele. In AD patients (n=28), CSF AChE activity correlated positively with CSF levels of T-tau and P-tau, but CSF activities of AChE or BuChE activity did not correlate with serum or CSF levels of 25OHD.Little is known of CSF 25OHD concentrations in human AD. In the present study, CSF 25OHD level was lower in AD patients compared to patients with other dementias and healthy controls. As previously observed in healthy individuals  and in mAll the included patients were community-dwelling and in the early phases of cognitive impairment/dementia with only a moderate reduction of MMSE score. Non-institutionalized older individuals have better vitamin D status compared to those living in homes for elderly as dietary intake of vitamin D and sun exposure are low in the latter group . In olde1-42, T-tau, and P-tau [In line with the results of a previous study , CSF BuCnd P-tau . FurtherCSF AChE activity was lower in the other dementia group compared to all other study groups. Some previous studies have also reported a more marked decrease of CSF AChE activity in VaD than in AD ,38. HoweAlthough this could have been due to limited statistical power, the distribution of the K-variant was similar in all study groups. Furthermore, in the total study population, CSF activities of AChE or BuChE were not dependent on BuChE K-variant distribution. Previous studies have shown conflicting results with an increased risk of late-onset AD in the presence of both the K-variant and the APOE ε4 allele in one study  but not In a previous study, AD patients treated with AChE inhibitors had higher vitamin D levels than non-treated patients . In our Different mechanisms of action in terms of vitamin D and AChE could be of clinical importance. Treatment of AD patients with AChE inhibitors have resulted in limited clinical effects. In terms of vitamin D, systematic reviews/meta-analyses of epidemiological data showed that lower circulating vitamin D concentrations were associated with poorer cognitive function and a higher risk of AD ,11. In aOne weakness of this study is the cross-sectional design, and changes over time in calcium/vitamin D metabolism and CSF activity of cholinesterases could therefore not be followed. Furthermore, this was a pilot study with a relatively small study population, and the results therefore need to be confirmed in further studies. Gender distribution was statistically similar in the study groups, but it can not be excluded that small differences in gender distribution could have been of importance as subanalyses showed lower serum 25OHD level in female but not in male AD patients compared to gender-matched SMCI patients and controls. In the group with other dementia, there were non-significant tendencies that BMI as well as the proportion of women were lower, and that the proportion of patients recruited during winter were higher, compared to the other study groups. Finally, BMI correlated positively with CSF BuChE activity both in the total study population and in AD patients and in addition, BMI correlated positively with CSF AChE activity in the total study population. Small, non-significant differences between groups in terms of body composition could therefore have influenced our results in terms of CSF activities of BuChE and AChE. In conclusion, in a homogenous, well-controlled study cohort, the CSF level of 25OHD was reduced in AD patients compared to patients with other dementias and healthy controls. The reduced CSF AChE activity correlated with CSF levels of AD biomarkers whereas CSF 25OHD level did not. Only CSF AChE activity was dependent on APOE genotype with the participants that were homozygous in terms of the APOE ε4 allele having decreased CSF AChE activity compared to subjects lacking the APOE ε4 allele. Furthermore, there was no correlation between levels of 25OHD and CSF activities of cholinesterases. Therefore, both 25OHD and AChE could be of importance for cognitive function in AD although possibly by different mechanisms of action. These findings might suggest that there is a rationale of combining vitamin D and AChE inhibitors in treatment trials.


Pythium insidiosum, which typically occurs in tropical and subtropical regions. The high mortality rate may be in consequence of the lack of diagnosis. The objective of this study was to evaluate reliability of a new single-tube nested PCR for detection of P. insidiosum DNA. A total of 78 clinical isolates of various fungi and bacteria, 106 clinical specimens and 80 simulated positive blood samples were tested. The developed primer pairs CPL6–CPR8 and YTL1–YTR1 are located on 18S subunit of the rRNA gene of P. insidiosum. The specificity, negative and positive predictive values were 100, 100 and 87.5 %, respectively, as compared with direct microscopy and cultivation. The detection limit of the single-tube nested PCR was 21 zoospores corresponding to 2.7 pg of the DNA. The results demonstrate that the new single-tube nested PCR offers a highly sensitive, specific and rapid genetic method for detecting P. insidiosum.Pythiosis is a rare infectious disease caused by  Pythium insidiosum, which can develop in humans and animals [P. insidiosum is a biflagellate zoospore [P. insidiosum can spread via the bloodstream to various internal organs or organ systems such as the gastrointestinal tract, brain, liver, kidney or rhinosinus [P. insidiosum in a single tube and to evaluate its reliability using various clinical specimens, including simulated positive blood samples and clinical isolates of bacteria and fungi.Pythiosis is an emerging and life-threatening infectious disease caused by the oomycete  animals –4. The izoospore . Human pzoospore , 5, 6. Tzoospore . Accordizoospore . Chroniczoospore . The ocuinosinus . Currentinosinus , 11–13. inosinus . Cultureinosinus . AlthougP. insidiosum as specified in Table Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Basidiobolus ranarum, Candida albicans, Candida tropicalis, Cladophialophora carrionii, Curvularia spp., Exophiala jeanselmei, Filobasidiella neoformans, Fusarium spp., Hortaea werneckii, Lichtheimia spp., Microsporum gypseum, Mucor spp., Penicillium spp., Rhodotorula spp., Rhizopus spp., Saccharomyces cerevisiae, Scedosporium apiospermum, Syncephalastrum spp., Talaromyces marneffei, Trichophyton spp., Trichophyton concentricum, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton violaceum and Trichosporon spp.), 10 bacterial isolates  and 5 isolates of non-insidiosum Pythium spp. . All microorganisms were obtained from patients of the Siriraj Hospital, Mahidol University, Bangkok, Thailand. All isolates were identified based on conventional microbiological methods and then stored in skim milk  100 mg/ml with 33 % glycerol at −20 °C.The study comprised 34 isolates of n = 27), tissue biopsies (n = 19), blood (n = 10), bone marrow (n = 9), lymph nodes (n = 6), peritoneal dialysis fluid (n = 6), sputum (n = 5), bronchial washing (n = 5), tracheal secretion (n = 5), urine (n = 5), cerebrospinal fluid (n = 4), synovial fluid (n = 2), ascitic fluid (n = 2) and pleural fluid (n = 1). In addition, 80 simulated positive blood specimens were prepared from normal blood samples. For this purpose, each blood sample was mixed with zoospores of P. insidiosum so that each sample contained 1.15 × 106 zoospores ml/l.One hundred and six clinical specimens from patients with suspected fungal infection obtained from a routine mycology laboratory in the Srinagarind Hospital, Khon Kaen University, Khon Kaen, Thailand were evaluated prospectively from May 2011 to February 2012. They included pus , two Mycosel agars  and one blood agar  for detection of P. insidiosum growth. One SDA and MCA each were incubated at 25 °C and the other media at 37 °C. All agars were evaluated for the P. insidiosum growth until 30 days. The suspected colonies were identified as P. insidiosum by induction of zoospores [All clinical specimens were evaluated microscopically in 20 % potassium hydroxide preparation for the presence of oospores . ResultsDNA from all clinical and simulated positive specimens was extracted with the NucleoSpin Tissue kit  and QIAamp DNA Mini Kit (Qiagen) according to the manufacturers’ instructions. All fungal isolates were cultured in 250 ml of Sabouraud dextrose broth  and incubated at a room temperature for 7 days with shaking (150 rpm) in a rotary shaker . Fungal mycelia were filtered, washed twice with deionized water and frozen at −20 °C until used. Bacterial isolates were cultured in 3 ml of Luria broth  and incubated at 37 °C for 16–18 h with shaking (200 rpm). Then, cultures were transferred to a 1.5 ml microtube, centrifuged and stored at 4 °C. For cell disruption, approximately 30 mg of frozen fungal mycelia and 0.14–1.21 g of the bacterial pellet were rubbed in liquid nitrogen until a fine powder. The bacterial powder was then suspended in a lysis buffer . DNA was extracted from the bacterial suspensions and powders from fungal mycelia as described by Sambrook and Russell [P. insidiosum (GenBank accession no. AF442497). The position of the newly designed primer is shown in Fig. 2, 25 pmol of each primers and 0.2 mM dNTPs. Amplification was performed in a thermal cycler  using the following temperature cycles: initial denaturation at 95 °C for 5 min, followed by 30 cycles , another 30 cycles  and final extension for 10 min at 72 °C. Amplicons were visualized under UV light using a manual documentation system  after electrophoresis of 10 μl of the reaction solution in 2 % UltraClean agarose gel (Mo Bio Laboratories) containing the SYBR Gold nucleic acid gel stain (Invitrogen).The single-tube nested PCR was performed using outer primers CPL6 (5′-GAC ACA GGG AGG TAG TGA CAA TAA ATA-3′) and CPR8 (5′-CTT GGT AAA TGC TTT CGC CT-3′), and inner primers YTL1 (5′-CTT TGA GTG TGT TGC TAG GAT G-3′) and YTR1 (5′-CTG GAA TAT GAA TAC CCC CAA C-3′) designed by ourselves using the GenBank database. The primers are located on the 18S subunit of the rRNA gene of 5–2.07 × 10−1 zoospores was used for nested PCR. As a control, DNA of P. insidiosum hyphae was also prepared as 10-fold serial dilution from 1 ng to 1 fg, and then, it was also amplified by a single-tube nested PCR. This study was approved by the Ethical Committee on Human Experimentation in Khon Kaen University in accordance with the Declaration of Helsinki (HE 541111).To determine the lowest detection limit of our nested PCR, seven 10-fold dilutions were prepared from simulated positive blood specimen. Then, 180 μl of each dilution containing 2.07 × 10P. insidiosum DNA was presented in clinical material, four amplicons sized 512, 452, 340 and 240 bp were detected by our nested PCR.If P. insidiosum isolates. Only one genus-specific product (512 bp) was found in five non-insidiosum Pythium spp. as demonstrated in Fig. Four amplicons were detected in all 34 roduct 51 bp was fBased on positivity of phenotypic methods, seven pus specimens from corneal ulcers were evaluated as positive within all tested clinical specimens. From that, three specimens were microscopically negative; culture and zoospore induction were positive in all seven specimens. Moreover, as demonstrated in Table P. insidiosum hyphae was 1 pg.As shown in Fig. P. insidiosum. Using our single-tube nested PCR, DNA of P. insidiosum was detected in 8 specimens, whereas 7 of them were positive by phenotypic methods. Therefore, NPV and PPV were 100 and 87.5 %, respectively.These results are summarized in Table P. insidiosum isolated from its mycelium was 1 pg of DNA, corresponding to about eight zoospores. According to this detection limit, one zoospore could contain 48–129 fg of DNA. This is the first report which tried to estimate the amount of DNA in P. insidiosum zoospores. Hussain et al. [Phytophthora infestans of 0.5 pg, which corresponded to four zoospores; in that case, one zoospore should contain 125 fg of DNA. The DNA content is similar to data about plant pathogenic organisms from the order Peronosporales (Oomycota) ranging from 46 to 163 fg [P. insidiosum and in clinical samples from patients with pythiosis. A sensitivity of 100 % and a low detection limit allow us to recommend our single-tube nested PCR as a useful tool for laboratories, which need to detect P. insidiosum in clinical material. Pythiosis is a rare but fatal infection in humans. Krajaejun et al. [P. insidiosum; therefore, the selected spiking of blood with zoospores simulates the state in really infected blood better than spiking it with pure DNA. The sensitivity of our nested PCR tested on 78 various clinical isolates and 186 clinical specimens including simulated positive blood samples was higher than with other tests [P. insidiosum directly from clinical specimens and identification of clinical isolates as well. As this method is rapid, highly sensitive and has minimal risk of cross-contamination, it has a great potential in the diagnosis of suspected cases of pythiosis.In our set of clinical specimens, one patient was found to be positive in single-tube nested PCR, but negative in phenotypic tests. In our study, the criterion for detecting pythiosis was positivity in one of two phenotypic methods. However, Krajaejun et al.  defined n et al.  reportedo 163 fg . The newer tests –22. In a


A microfiber is used as a laser-focusing unit to fabricate a groove structure on TiAlSiN surfaces. After one laser pulse etching, a groove with the minimum width of 265 nm is manufactured at the area. This technique of microfabricating the groove in microscale is studied. Based on the near-field intensity enhancement at the contact area between the fiber and the surface during the laser irradiation, simulation results are also presented, which agree well with the experimental results. In the past 10 years, with the important role of microdevices in industrial applications, fabrication technologies on the micron and nanometer scales are attracting more attentions. Recently, developed lithographic technologies like ion beam, electron beam, and near-field optics have been investigated and regarded as potential methods for microfabrication. Due to A laser etching experiment with the microfiber-enhanced irradiation is designed to fabricate a groove on TiAlSiN surfaces Figure. Nd: YAG2. The laser perpendicularly irradiates on TiAlSiN through the fiber and etches the surface. After one laser pulse etching, a groove on the TiAlSiN surface is created. It is observed under both SEM and atomic force microscopy (AFM).In the groove manufacture process, the micro-fiber is placed on the TiAlSiN surface. The fiber contacts with the surface directly, which makes the gap between the fiber and the surface at nanometer scales. Light intensity at the sample surface is adjusted at 440 mJ/cmRF module of Comsol 3.5 is used to simulate optical fields. Here, it is required to calculate the energy distribution behind the fiber. The calculations are based on finite element analysis to solve Maxwell equations with the boundary conditions. The calculation results are compared to the groove characters.2, as shown in FigureFigureThis etching technique of the microfiber-enhanced irradiation is also attemped to process grooves on Si. A inhomogeneous groove with a minimum size of around 300 nm is also observed on the laser treated surface. As the fiber changes in the diameters, the groove dimension is relatively altered. However, the further investment needs to find out the fabrication technique and etching mechanism.FigureCalculations of the light intensity under the fiber are carried out by solving the electromagnetic boundary problem through ’Maxwell equations’. Here, fiber models in Comsol 3.5 permit us to estimate the groove characters. FigureTwo mechanisms of Rayleigh scattering and Mie scattering are proposed to explain the enhancement by the laser irradiation of particles. The intA novel laser etching technique, in which a quartz fiber at a diameter of about 5 μm is placed on the TiAlSiN surface in order to induce the light intensity enhancement near the contact line, is investigated. We have demonstrated that the laser etching with microfiber-enhanced irradiation can be used to fabricate a groove. Its minimum width is about 265 nm. This technique is found to have advantages of simple setup and low cost. Meanwhile, the experimental results are consistent with computational simulations.The authors declare that they have no competing interests.DL carried out the design and fabrication of the experimental setups and drafted the manuscript. JL participated in the fabrication of the grooves. WH established the theoretical formalism and participated in the numerical calculations. TS supervised the whole study. All authors read and approved the final manuscript.DL is a lecturer in State Key Laboratory of Tribology, Tsinghua University. JL is a PHD student. HW is a research assistant. TS is a professor of State Key Laboratory of Tribology, Tsinghua University.


During the early stages of brain development, ependymal cells that line the neural tube are thought to secrete CSF. It is well documented that fetal CSF contains many neurotrophic and growth factors which are known as modulators of neurogenesis, differentiation and brain extracellular microenvironment. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since the cells undergo differentiation to sympathetic neuron-like cells in response to NGF, bFGF, EGF, TGF-α and GDNF. We hypothesized that prenatal CSF could have differentiational effect on PC12 cells.CSF was removed by tapping the cisterna magna of Wistar rat fetuses (E17- E20) then centrifuged at 4000 r.p.m for 10 min, the supernatant frozen immediately and stored at -70°C until used. PC12 cells were cultured in RPMI-1640 with 10% FBS, 100 unit/ml of penicillin, 100 mg/ml of streptomycin and 5% CO2 at 37°C. For culture experiments, 2*10E4 PC12 cells were added to each well of a 96-well plate that had been coated with Poly-D-Lysine. After attachment, the cells were exposed to CSF at different ages with dissimilar concentration of 7, 10, 25% (v/v). The cell viability and cell proliferation were measured by MTT assay. The neuronal differentiation of PC12 cells were considered by changes of neurite outgrowth.Viability and cell proliferation were significantly elevated in PC12 cells cultured in CSF supplemented medium in E18 compared with control ones. A significant neuronal-like outgrowth appeared as early as Day 3 after the application of the CSF supplemented medium E17.It was shown that CSF neurotrophic factors can support normal neurogenesis and promotes proper brain development, neuronal differentiation. It has been reported that CSF can be a survival material on its own with any medium for cerebral cortex primary cultures. Our data are in the same line with pervious studies that clarify crucial role of CSF neurotrophic factors in neuronal differentiation and cell proliferation. Taken together we address PC12 neuronal differentiation and cell proliferation to CSF induction by its components especially growth factors.