Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls (n = 14). miRNAs were quantified by RT-qPCR, normalized to "spike-in" cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH.

Pitfalls in the diagnosis of meningitis in neonates and young infants: the role of lumbar puncture.

Meningitis occurs frequently in neonates and can lead to a number of acute, severe complications and long-term disabilities. An early diagnosis of neonatal meningitis is essential to reduce mortality and to improve outcomes. Initial clinical signs of meningitis are often subtle and frequently overlap with those of sepsis, and current haematologic tests do not distinguish sepsis from meningitis. Thus, lumbar puncture (LP) remains the gold standard for the diagnosis of meningitis in infants, and this procedure is recommended in clinical guidelines. Nevertheless, in clinical practice, LP is frequently deferred or omitted due to concerns regarding hypothetical adverse events or limited experience of the performer. Future studies should assess whether a combination of clinical findings and select haematologic tests at disease onset can identify those neonates with the highest risk of meningitis who should undergo LP. Furthermore, clinicians should be convinced that the actual benefits of an early diagnosis of meningitis far outweigh the hypothetical risks associated with LP.

The microbial spectrum of neonatal sepsis in Uganda: recovery of culturable bacteria in mother-infant pairs.

Neonatal sepsis in the developing world is incompletely characterized. We seek to characterize the microbial spectrum involved in sepsis and determine the role of maternal transmission by comparing organisms that can be cultured from septic newborn infants and their mothers. From 80 consecutive mother-infant pairs meeting clinical criteria for neonatal sepsis, we collected infant blood and spinal fluid, and maternal blood and vaginal specimens. Identifiable bacteria were recovered from the blood in 32.5% of infants, and from 2.5% of cerebrospinal fluid cultures, for a total of 35% recoverable putative causative agents. Bacteria recovered from vaginal specimens were not concordant with those recovered from infants. Similarly there was no concordance of bacteria recovered from blood and cerebrospinal fluid. We conclude that relying on traditional bacterial culture techniques does not adequately delineate the role of maternal versus environmental sources of neonatal sepsis in this setting. More sensitive molecular approaches will be needed to properly characterize the maternal and environmental microbial community involved in neonatal sepsis in such developing countries.

Detection of cerebral spinal fluid-associated chemokines in birth traumatized premature babies by chip-based immunoaffinity CE.

A major concern in treating premature infants with birth-associated head trauma is the rapid determination of reliable biomarkers of neuroinflammation. To this end a chip-based immunoaffinity CE device has been applied to determine the concentrations of inflammation-associated chemokines in samples of cerebral spinal fluid collected from such subjects. The chip utilizes replaceable immunoaffinity disks, to which reactive antibody fragments (FAb) of six antichemokine-specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labeled in situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on-line by LIF detection and the results compared to standard curves produced by running known chemokine standards through the immunoaffinity system. The complete processing of a sample took 10 min with separation of all six analytes being achieved in less than 2 min. The system compared well to commercial ELISA, analysis of the results by linear regression demonstrating r(2) values in the range of 0.903-0.978, and intra and interassay CV of the migration times and the measured peak areas being less than 2.3 and 5%, respectively. Application of the system to analysis of cerebrospinal fluid from head traumatized babies clearly indicated the group with mild trauma versus those with severe injury. Additionally, CE analysis demonstrated that the severe trauma group could be divided into individuals with good and poor prognosis, which correlated with the clinical finding for each patient.

Increased expression of matrix metalloproteinase-9 and hepatocyte growth factor in the cerebrospinal fluid of infants with posthemorrhagic hydrocephalus.

BACKGROUND: In approximately 60% of infants with posthemorrhagic hydrocephalus (PHH), ventricular dilation resolves by unknown intrinsic mechanisms, without the need for a shunt operation. A pathological hallmark of PHH is extensive deposition of extracellular matrix (ECM) proteins in the subarachnoid space. Our previous study revealed that matrix metalloproteinase (MMP)-9, which degrades ECM proteins, may play an important role in the resolution of ventricular dilation. MMP-9 is known to be induced by hepatocyte growth factor (HGF) in various cell lines. AIMS: The aim of this study is to confirm our earlier finding that MMP-9 contributes to the resolution of PHH, and to investigate whether HGF also contributes to this process. STUDY DESIGN: Cerebrospinal fluid (CSF) samples were collected from 13 infants who developed ventricular dilation after intraventricular hemorrhage (IVH). Of these infants, 9 exhibited resolution of ventricular dilation without shunt operation; however, 4 infants had to be treated with shunt operation. The CSF levels of MMP-9 and HGF were measured using an enzyme immunoassay. RESULTS: Significantly higher CSF levels of MMP-9 and HGF were detected in patients in whom the ventricular dilation resolved without shunt operation than in those with progressive ventricular dilation (MMP-9: median, 128ng/ml; range, 47-900ng/ml vs median, 50ng/ml; range, 12-110ng/ml; p<0.05; HGF: median, 2.42ng/ml; range, 0.81-7.04ng/ml vs median, 1.42ng/ml; range, 0.67-3.87ng/ml; p<0.05). CONCLUSIONS: Our results indicate that MMP-9 and HGF may participate in the resolution of ventricular dilation following IVH.

Cerebrospinal fluid leucocyte counts in healthy neonates.

OBJECTIVES: To determine the cerebrospinal fluid (CSF) white blood cell (WBC) count of normal term neonates, and compare the CSF WBC profile of normal and symptomatic infants without infection of the central nervous system (CNS). METHOD: Neonates were included if (a) they were at risk of congenital Toxoplasma infection and had undergone a lumbar puncture to assess CNS involvement, and (b) serial specific serum IgG and IgM determinations had ruled out congenital infection. According to neonatal chart reviews, 30 consecutive patients without CNS infection were classified as normal (absolutely asymptomatic) or symptomatic (any kind of symptoms). RESULTS: CSF WBC count was higher in 11 symptomatic (7/mm(3), 0-30/mm(3)) than in 19 normal (1/mm(3), 0-5/mm(3)) neonates (p<0.01). CONCLUSION: Normal neonatal CSF contains up to 5 WBCs/mm(3). Mild pleocytosis can be found in symptomatic infants without CNS infection.

Electrolyte and glucose concentration in plasma and cerebrospinal fluid measured parallel in pathologic newborn infants.

Cerebrospinal fluid and plasma sodium, potassium, chloride, calcium and glucose concentration were measured parallel in 14 pathological newborn babies of gestational age and birthweight of 36.3 +/- 4.3 wks and 2410 +/- 890 g, respectively, at the age of 37.8 +/- 4.4 wks postconceptionally. Whilst potassium, calcium and glucose level is much lower in the cerebrospinal fluid than in the plasma, similar sodium and higher chloride concentration was found in the cerebrospinal fluid. The significant positive correlation between plasma and cerebrospinal fluid glucose and sodium levels proves the lack of a functioning barrier for these compounds. On the other hand, cerebrospinal potassium level varied within a narrow range, independently of the plasma-concentration and the maturity of the studied babies. Pathophysiological implications of the results are further discussed in short.

[Interrelation of bilirubin and free fatty acids in newborn infants with pathologic conditions]. / Interrelaciones entre bilirrubina y ácidos grasos libres en recién nacidos patológicos.

Authors analyzed total and free bilirubin concentrations (TB and FB) and free fatty acid levels (FFA) (by enzymatic methods) in the serum (S) and cerebrospinal fluid (CSF) of 26 sick newborn infants. Relationship between these three biochemical parameters in both S and CSF and also between these latter 15 studies FFA serum concentration of neonates (n: 9) with TB less than 2 mg/dl - 70.15 (15.63) (+/- SEM) mg/l - was not statistically different from those calculated in CSF - 67.45 (13.80) mg/l - (p: NS): while newborn infants (n: 17) with TB greater than 2 mg/dl had FFA serum levels - 152.75 (22.84) less than 0.002). There was a positive correlation between TB and FFA in S (n: 26, r: 0.52, p less than 0.01). There was no correlation of the FFA levels between S and CSF (n: 26, r: -0.02, p: NS). In CSF, FFA concentrations were correlated with albumin levels (n: 26, r: 0.67, p less than 0.005) and white cell recound (n: 26, r: 0.53, p less than 0.01). Authors discuss results and conclude that: a) bilirubin interferes with FFA metabolism, probably as the result of a toxic effect on extraneuronal tissue; b) an increase of FFA serum concentrations does not cause them to rise in the CSF by transport across blood cerebrospinal fluid barrier; and c) although, the source of the FFA in CSF is varied (from serum or loss of myelin, for instance, as a consequence of an asphyxial insult, or devitalized white cells) their increased concentrations are correlated with enhanced albumin levels and white cell recounts.

Neurotransmitter precursors and metabolites in CSF of human neonates.

The amino-acid precursors tryptophan and tyrosine, and the major metabolites 5-hydroxyindoleacetic acid, indoleacetic acid, homovanillic acid and 3-methoxy-4-hydroxyphenethyleneglycol, related to the central neurotransmitters serotonin, dopamine and norepinephrine, were measured in 62 samples of cerebrospinal fluid from human neonates. Means are reported for the samples from 17 medically uncomplicated infants and for the larger group (42 to 45) of infants with medical complications. The latter group was divided according to diagnosis and medication. All groups had significantly higher levels of all compounds in comparison with older children and adults. There were few significant subgroup differences in the group with complications. In both the normal and complicated groups a number of significant correlations were observed between the compounds themselves and with other physiological measures.

Ventricular configuration and cerebral growth in infants born to drug-dependent mothers.

Cranial ultrasound examinations were performed during the first 3 days of life and at age 1 month on 22 infants with the neonatal abstinence syndrome. The results were compared to those obtained in 15 control infants who were not exposed to narcotic drugs in utero. The ultrasound images were examined for ventricular configuration, intracranial hemidiameters, area of thalami, and width of temporal lobes. At 24 to 72 h and at 1 month of age, significantly more drug-exposed than control infants had a slit-like ventricular configuration. The intracranial hemidiameter was significantly smaller in the drug-exposed than in the control infants. All cerebral measurements except the right temporal lobe demonstrated significant growth over the first month of life in both groups of infants. By means of ancillary examinations (computerized tomography and transfontanel pressure measurements) the pathogenesis of the slit-like ventricles was found not to be related to edema or to increased intracranial pressure. Whether or not the ventricles remain small and brain growth remains parallel after the period of abstinence awaits further investigation.

Bacterial meningitis. Effect of antibiotic treatment on cerebrospinal fluid.

The effects of full short-term antibiotic treatment on cerebrospinal fluid (CSF) findings were studied retrospectively in 68 children with acute bacterial meningitis. The features of CSF at admission were compared with those of the CSF obtained after 44-68 hours of therapy. Except in one case with H. influenzae and one case with pneumococcal meningitis, all CSF cultures were negative in the repeat specimen. In three of 16 children with meningococcal meningitis, the CSF glucose levels became normal in the second specimen. In all remaining 65 children, however, full intravenous antibiotic treatment for 44-68 hours did not alter the biochemistry and cytology of the CSF, which retained its "bacterial" character. From these findings it may be discerned that partial antibiotic treatment is even less likely to distort a 'bacterial' CSF.

Systemic bacterial infections in neonatal deaths.

Bacterial were identified in 126 blood and CSF cultures obtained in 311 consecutive neonatal deaths (41%). These postmortem cultures were of diagnostic value, providing the sole means for definitive bacteriologic diagnosis in 82 (65%) of the 126 infected infants. Similarity of organisms found in specimens before and after death (identical in 25 of 26), similar identity of organisms identified by histologic Gram's stain and culture (the same in 48 of 49), and the identical nature of organisms identified from blood and CSF sites (the same in 43 of 43) support the validity of these cultures. Bacterial infection remains a serious problem in neonatal intensive care. The scope of this problem may be underestimated if postmortem cultures are not obtained.

Elevation of cAMP levels in cerebrospinal fluid of patients with neonatal meningitis.

The cyclic 3',5'-adenosine monophosphate (cAMP) concentrations in the cerebrospinal fluid of 20 children with neonatal bacterial meningitis and aseptic meningitis were measured by radioimmunoassay method. The cAMP levels were found to be significantly elevated above control levels (P less than .01) during the acute phase in most of the patients. In the convalescent stage the cAMP concentration was decreased but levels remained significantly elevated (P less than .01) in patients with complications. During the acute phase, the cAMP levels were higher in neonatal bacterial meningitis than in aseptic meningitis (P less than .01). The results suggest that cAMP is a sensitive indicator of transient cellular metabolic disturbance in the brain and may be used to monitor the course of neonatal meningitis.

gamma-Aminobutyric acid concentrations in the cerebrospinal fluid of newborn infants.

gamma-Aminobutyric acid (GABA) concentrations in cerebrospinal fluid (CSF) were measured in 20 neonates with various gestational and postnatal ages. These concentrations varied between 9 and 45 nmol/ml which is approximately 20-100 times the concentrations found in adults. CSF GABA concentrations tended to decrease with advancing gestational age. No apparent alterations were noted with increasing postnatal age (until 6 weeks of age). Asphyxia but not neonatal sepsis was accompanied by an increase in CSF GABA concentrations compared to respective controls.

Cerebrospinal fluid acid-base balance in newborns.

In adults, cerebrospinal fluid (CSF) acid-base balance is an important determinant of cerebral blood flow, ventilatory drive, and state of consciousness. Sixteen lumbar punctures were performed on newborns (mean gestational age, 33.8 weeks; range, 26 to 44 weeks). The mean CSF pH was 7.366 units, 0.036 less than the mean capillary blood pH (NS). The mean CSF arterial carbon dioxide tension (PCO2) was 40.3 mm Hg, 6.7 greater than the mean blood PCO2 (p less than 0.01). The mean bicarbonate ion concentration in CSF was 21.8 mEq/L, 1.2 greater than that in blood (NS). Acid-base determinations in CSF of neonates with intraventricular hemorrhage did not differ significantly from those of neonates with otherwise normal CSF. These acid-base values are similar to those found in adults and did not vary with gestational age, suggesting that mechanisms to establish this acid-base gradient are developed by 26 weeks' gestation.