Xenon combined with therapeutic hypothermia is not neuroprotective after severe hypoxia-ischemia in neonatal rats

Therapeutic hypothermia (TH) is standard treatment following perinatal asphyxia in newborn infants. Experimentally, TH is neuroprotective after moderate hypoxia-ischemia (HI) in seven-day-old (P7) rats. However, TH is not neuroprotective after severe HI. After a moderate HI insult in newborn brain injury models, the anesthetic gas xenon (Xe) doubles TH neuroprotection. The aim of this study was to examine whether combining Xe and TH is neuroprotective as applied in a P7 rat model of severe HI.120 P7 rat pups underwent a severe HI insult; unilateral carotid artery ligation followed by hypoxia (8% O2 for 150min at experimental normothermia (NT-37: Trectal 37°C). Surviving pups were randomised to immediate NT-37 for 5h (n = 36), immediate TH-32: Trectal 32°C for 5h (n = 25) or immediate TH-32 plus 50% inhaled Xe for 5h (n = 24). Pups were sacrificed after one week of survival. Relative area loss of the ligated hemisphere was measured, and neurons in the subventricular zone of this injured hemisphere were counted, to quantify brain damage.Following the HI insult, median (interquartile range, IQR) hemispheric brain area loss was similar in all groups: 63.5% (55.5-75.0) for NT-37 group, 65.0% (57.0-65.0) for TH-32 group, and 66.5% (59.0-72.0) for TH-32+Xe50% group (not significant). Correspondingly, there was no difference in neuronal cell count (NeuN marker) in the subventricular zone across the three treatment groups.Immediate therapeutic hypothermia with or without additional 50% inhaled Xe, does not provide neuroprotection one week after severe HI brain injury in the P7 neonatal rat. This model aims to mimic the clinical situation in severely asphyxiated neonates and treatment these newborns remains an ongoing challenge

A time-resolved fluorescence immunoassay for the measurement of testosterone in saliva: Monitoring of testosterone replacement therapy with testosterone buciclate

Monitoring of testosterone replacement therapy requires a reliable method for testosterone measurement. Determination of salivary testosterone, which reflects the hormone's biologically active plasma fraction, is a superior technique for this purpose. The aim of the present study was to establish a new sensitive time-resolved fluorescence immunoassay for the accurate measurement of testosterone levels in saliva and to validate it by monitoring testosterone replacement therapy in eight hypogonadal men. A clinical phase I- study with the new ester testosterone buciclate was performed to search for new testosterone preparations to produce constant serum levels in the therapy of male hypogonadism. After two control examinations eight male patients with primary hypogonadism were randomly assigned to two treatment groups (n = 2x4) and given single doses of either 200 mg (group I) or 600 mg (group II) testosterone buciclate intramuscularly. Saliva and blood samples were obtained 1, 2, 3, 5 and 7 days post injection and then weekly for three months. The time-resolved fluorescence immunoassay for salivary testosterone shows a detection limit of 16 pmol/l, an intra-assay CV of 8.9% (at a testosterone concentration of 302 pmol/l), an inter-assay CV of 8.7% (at a testosterone concentration of 305 pmol/l) and a good correlation with an established radioimmunsassay of r = 0.89. The sample volume required by this method is only 180 mu l for extraction and duplicate determination. The assay procedure requires no more than three hours. In group I (200 mg) testosterone did not increase to normal levels either in saliva or in serum. However, in group II, androgen levels increased significantly and were maintained in the normal range for up to 12 weeks with maximal salivary testosterone levels of 303 +/- 18 pmol/l (mean+/-SE) and maximal testosterone levels of 13.1 +/- 0.9 nmol/l (mean+/-SE) in serum in study week 6 and 7. The time-resolved fluorescence immunoassay for salivary testosterone provides a useful tool for monitoring androgen status in men and women and is well suited for the follow-up of testosterone replacement therapy on an outpatient basis. The long-acting ester testosterone buciclate is a promising agent for substitution therapy of male hypogonadism and in combination with testosterone monitoring in saliva offers an interesting new perspective for male contraception

Variability and sex-dependence of hypothermic neuroprotection in a rat model of neonatal hypoxic-ischaemic brain injury:a single laboratory meta-analysis

Therapeutic hypothermia (HT) is standard care for term infants with hypoxic–ischaemic (HI) encephalopathy. However, the efficacy of HT in preclinical models, such as the Vannucci model of unilateral HI in the newborn rat, is often greater than that reported from clinical trials. Here, we report a meta-analysis of data from every experiment in a single laboratory, including pilot data, examining the effect of HT in the Vannucci model. Across 21 experiments using 106 litters, median (95% CI) hemispheric area loss was 50.1% (46.0–51.9%; n = 305) in the normothermia group, and 41.3% (35.1–44.9%; n = 317) in the HT group, with a bimodal injury distribution. Median neuroprotection by HT was 17.6% (6.8–28.3%), including in severe injury, but was highly-variable across experiments. Neuroprotection was significant in females (p < 0.001), with a non-significant benefit in males (p = 0.07). Animals representing the median injury in each group within each litter (n = 277, 44.5%) were also analysed using formal neuropathology, which showed neuroprotection by HT throughout the brain, particularly in females. Our results suggest an inherent variability and sex-dependence of the neuroprotective response to HT, with the majority of studies in the Vannucci model vastly underpowered to detect true treatment effects due to the distribution of injury

Case Report: SATB2-Associated Syndrome Overlapping With Clinical Mitochondrial Disease Presentation: Report of Two Cases

SATB2-associated syndrome (SAS) is an autosomal dominant neurogenetic multisystemic disorder. We describe two individuals with global developmental delay and hypotonia who underwent an extensive evaluation to rule out an underlying mitochondrial disorder before their eventual diagnosis of SAS. Although the strict application of the clinical mitochondrial disease score only led to the designation of “possible” mitochondrial disorder for these two individuals, other documented abnormalities included nonspecific neuroimaging findings on magnetic resonance imaging and magnetic resonance spectroscopy, decreased complex I activity on muscle biopsy for patient 2, and variation in the size and relative proportion of types of muscle fibers in the muscle biopsies that were aligned with mitochondrial diseases. SAS should be in the differential diagnoses of mitochondrial disorders, and broad-spectrum diagnostic tests such as exome sequencing need to be considered early in the evaluation process of undiagnosed neurodevelopmental disorders

Minor neurological signs and behavioural function at age 2 years in neonatal hypoxic ischaemic encephalopathy (HIE)

Background: Neurodevelopmental follow-up in Neonatal Hypoxic Ischaemic Encephalopathy (HIE) typically focusses on major neuromotor (cerebral palsy, CP) and severe cognitive impairment. Outcomes in those without major neuromotor impairment are less well explored. Objectives: To examine behavioural, cognitive and neurological outcomes after neonatal HIE, in a clinical cohort of children without CP, at age 2 years. Methods: Clinical routine outcome data from children admitted to a tertiary centre with neonatal HIE for hypothermia treatment between 05/08/09 - 30/05/2016. Children were assessed for neuromotor status – particularly minor neurological signs (MNS), with Bayley Scales of Infant and Toddler Development III (Bayley III) or Ages and Stages Questionnaire-3 (ASQ), Child Behavior Checklist 1.5-5 (CBCL), Quantitative Checklist for Autism in Toddlers (Q-CHAT). Results: Of 107 children, 75.5% had normal neurology, 12.1% CP, 12.1% MNS. Children with CP were excluded from analyses. For those without CP, Bayley-III scores were in the average range for the majority; mild cognitive delay observed in 5%, 4.2% language, 1.3% motor development; severe delay in 1.3% for cognitive, 4.2% for language. More than in the normative population scored in clinical ranges for CBCL externalising, sleep, and other problems. No significant difference was seen for Q-CHAT. Children with MNS were significantly more likely to have impaired Bayley-III scores, parent-reported internalising, sleep, and other problems. Conclusions: In this clinical cohort, the majority of children had favourable outcome at 2 years. However, children with MNS were at risk for cognitive and behavioural difficulties and will benefit from enhanced clinical follow-up and support

Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia.

BACKGROUND: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. METHODS: Sixteen piglets were randomized: (i) LPS 2 μg/kg bolus; 1 μg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. RESULTS: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h. CONCLUSIONS: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE. IMPACT: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes