Protective Effects of Early Neonatal Methylxanthine Treatment on Cognitive and Language Outcomes in Premature Infants with and without High-Risk Perinatal Factors.

INTRODUCTION: Caffeine and theophylline are methylxanthines and nonselective adenosine antagonists commonly used to treat apnea of prematurity. Both human and animal data suggest that xanthines also have clinically important long-term neuroprotective effects in the presence of inflammation in the perinatal period as seen following hypoxic-ischemic brain insults. Moreover, these protective effects appear to be more robust when administered shortly (<48 h) after preterm birth. METHOD: To evaluate the importance of the postdelivery therapeutic window, we collected and analyzed medical data from preterm infants meeting criteria (23-30 weeks' gestational age [GA]), born at the University of Connecticut Health Center (UCHC), and cared for at the UCHC/Connecticut Children's Medical Center (CCMC) NICU from 1991 to 2017 (n = 858). Eighteen-month follow-up data included cognitive and language scores from the Neonatal Neurodevelopmental Follow-Up Clinic records, with a retention of 81% of subjects (n = 696). Differences were analyzed via multivariate ANOVA and ANCOVA. RESULTS: Analyses showed that infants who received xanthine treatment within the first 48 h after preterm birth showed significantly better 18-month behavioral outcomes than those treated later than 48 h, despite a lack of a priori differences in GA, birth, or length of stay. The positive effect of early xanthine therapy was particularly robust for infants exposed prenatally to the inflammatory conditions of chorioamnionitis and/or preeclampsia. CONCLUSIONS: Current findings are consistent with human and animal data, showing that caffeine exerts protective effects, at least in part via attenuation of inflammation. Results add to the evidence supporting routine immediate prophylactic neuroprotective xanthine therapy (i.e., caffeine) in preterm infants. Findings also add important new evidence of the augmented value of caffeine for infants with inflammatory exposure due to mothers with preeclampsia or chorioamnionitis.

A novel approach to the assessment of higher-order rule learning in male mice.

Historically, the development of valid and reliable methods for assessing higher-order cognitive abilities (e.g., rule learning and transfer) has been difficult in rodent models. To date, limited evidence supports the existence of higher cognitive abilities such as rule generation and complex decision-making in mice, rats, and rabbits. To this end, we sought to develop a task that would require mice to learn and transfer a rule. We trained mice to visually discriminate a series of images (image set, six total) of increasing complexity following three stages: (1) learn a visual target, (2) learn a rule (ignore any new images around the target), and finally (3) apply this rule in abstract form to a comparable but new image set. To evaluate learning for each stage, we measured (1) days (and performance by day) to discriminate the original target at criterion, (2) days (and performance by day) to get back to criterion when images in the set were altered by the introduction of distractors (rule learning), and (3) overall days (and performance by day) to criterion when experienced versus naïve cohorts of mice were tested on the same image set (rule transfer). Twenty-seven wild-type male C57 mice were tested using Bussey-Saksida touchscreen operant conditioning boxes (Lafayette Instruments). Two comparable black-white image sets were delivered sequentially (counterbalanced for order) to two identical cohorts of mice. Results showed that all mice were able to effectively learn their initial target image and could recall it >80 d later. We also found that mice were able to quickly learn and apply a "rule" : Ignore new distractors and continue to identify their visual target embedded in more complex images. The presence of rule learning was supported because performance criterion thresholds were regained much faster than initial learning when distractors were introduced. On the other hand, mice appeared unable to transfer this rule to a new set of stimuli. This is supported because visual discrimination curves for a new image set were no better than an initial (naïve) learning by a matched cohort of mice. Overall results have important implications for phenotyping research and particularly for the modeling of complex disorders in mice.

Antenatal Magnesium Sulfate Benefits Female Preterm Infants but Results in Poor Male Outcomes.

Magnesium sulfate (MagSul) is used clinically to prevent eclamptic seizures during pregnancy and as a tocolytic for preterm labor. More recently, it has been implicated as offering neural protection in utero for at-risk infants. However, evidence is mixed. Some studies found that MagSul reduced the incidence of cerebral palsy (CP) but did not improve other measures of neurologic function. Others did not find any improvement in outcomes. Inconsistencies in the literature may reflect the fact that sex effects are largely ignored, despite evidence that MagSul shows sex effects in animal models of neonatal brain injury. The current study used retrospective infant data to assess differences in developmental outcomes as a function of sex and MagSul treatment. We found that on 18-month neurodevelopmental cognitive and language measures, preterm males treated with MagSul (n = 209) had significantly worse scores than their untreated counterparts (n = 135; p < 0.05). Female preterm infants treated with MagSul (n = 220), on the other hand, showed a cognitive benefit relative to untreated females (n = 123; p < 0.05). No significant effects of MagSul were seen among females on language (p > 0.05). These results have tremendous implications for risk-benefit considerations in the ongoing use of MagSul and may explain why benefits have been hard to identify in clinical trials when sex is not considered.

Sex Differences in Microglia Activation in a Rodent Model of Preterm Hypoxic Ischemic Injury with Caffeine Treatment.

Preterm infants are often treated with caffeine as a respiratory stimulant. However, follow-up data shows caffeine may also have neuroprotective potential. There are several theories as to how caffeine might protect the brain, but none have been proven. This study looked at caffeine effects on microglial activation in rodent brains post hypoxic ischemic (HI) injury. Rat pups underwent either sham or HI surgery on P6, followed by treatment with either caffeine or saline. Forty-eight hours post-injury, brains were collected and underwent paraffin embedding and sectioning followed by immunofluorescence staining. Ionized calcium binding adaptor molecule 1 (Iba-1) was used to label microglia, and 4',6-diamindino-2-phenylindole (DAPI) was used to label DNA. Cell size measurements of microglia were obtained to gauge microglia activation, and chromatin condensation (DAPI optical density) was used as an index of neuronal cell death. Results suggest that caffeine does offer protective effects, based on significantly increased levels of cell death in HI-saline animals not seen in caffeine-treated HI males and females. However, the mechanism of action may be different. Male HI animals showed marginally reduced microglial activation following caffeine treatment, whereas females did not. Results indicate that though caffeine may act protectively in both sexes by reducing cell death, the benefits may be mediated by different mechanisms.

Therapeutic Interventions in Rat Models of Preterm Hypoxic Ischemic Injury: Effects of Hypothermia, Caffeine, and the Influence of Sex.

Infants born prematurely have an increased risk of experiencing brain injury, specifically injury caused by Hypoxia Ischemia (HI). There is no approved treatment for preterm infants, in contrast to term infants that experience Hypoxic Ischemic Encephalopathy (HIE) and can be treated with hypothermia. Given this increased risk and lack of approved treatment, it is imperative to explore and model potential treatments in animal models of preterm injury. Hypothermia is one potential treatment, though cooling to current clinical standards has been found to be detrimental for preterm infants. However, mild hypothermia may prove useful. Caffeine is another treatment that is already used in preterm infants to treat apnea of prematurity, and has shown neuroprotective effects. Both of these treatments show sex differences in behavioral outcomes and neuroprotective effects, which are critical to explore when working to translate from animal to human. The effects and research history of hypothermia, caffeine and how sex affects these treatment outcomes will be explored further in this review article.

Magnetic resonance cholangiopancreatography uncovering massive gallbladder mucocele in a patient with ambiguous clinical and laboratory findings: A case report.

INTRODUCTION: Radiological imaging of the gallbladder is a fundamental aspect of assessing severity of acute cholecystitis in addition to guiding the optimal timing of surgical intervention. We present a case of acute severe cholecystitis with massive gallbladder mucocele initially presenting with equivocal and inconclusive clinical, laboratory, and ultrasound findings. Magnetic resonance cholangiopancreatography allowed accurate evaluation of cholelithiasis and demonstrated a massive gallbladder mucocele. CASE PRESENTATION: A 39-year-old Caucasian female presented with mild right upper quadrant pain coupled with intermittent epigastric discomfort after meals. Diagnostic abdominal ultrasound could not reliably detect cystic or common bile duct stones due to patient obesity and meteorism. Computed tomography was contraindicated due to severe contrast allergy. Magnetic resonance cholangiopancreatography allowed timely, accurate evaluation of cholelithiasis. This subsequently demonstrated a massive gallbladder mucocele requiring urgent laparoscopic cholecystectomy. CONCLUSION: Magnetic resonance cholangiopancreatography should be considered as a complementary imaging modality to assess patients presenting with atypical biliary symptoms, particularly when ultrasound is equivocal or inconclusive, clinical and laboratory findings are non-specific, and computer tomography is contraindicated. Magnetic resonance cholangiopancreatography can also be considered in patients with acute cholecystitis not adherent to a specific severity grade.

p62 (SQSTM1) and cyclic AMP phosphodiesterase-4A4 (PDE4A4) locate to a novel, reversible protein aggregate with links to autophagy and proteasome degradation pathways.

Chronic challenge of cyclic AMP phosphodiesterase-4A4 (PDE4A4) with certain PDE4 selective inhibitors causes it to reversibly form intracellular aggregates that are not membrane-encapsulated. These aggregates are neither stress granules (SGs) nor processing bodies (PBs) as they contain neither PABP-1 nor Dcp1a, respectively. However, the PDE4 inhibitor rolipram decreases arsenite-induced SGs and increases the amount of PBs, while arsenite challenge ablates rolipram-induced PDE4A4 aggregates. PDE4A4 aggregates are neither autophagic vesicles (autophagosomes) nor aggresomes, although microtubule disruptors ablate PDE4A4 aggregate formation. PDE4A4 constitutively co-immunoprecipitates with p62 protein (sequestosome1, SQSTM1), which locates to both PDE4A4 aggregates and autophagosomes in cells constitutively challenged with rolipram. The mTor inhibitor, rapamycin, activates autophagy, prevents PDE4A4 from forming intracellular aggregates and triggers the loss of bound p62 from PDE4A4. siRNA-mediated knockdown of p62 attenuates PDE4A4 aggregate formation. The p62-binding protein, light chain 3 (LC3), is not found in PDE4A4 aggregates. Blockade of proteasome activity and activation of autophagy with MG132 both increases the level of ubiquitinated proteins found associated with PDE4A4 and inhibits PDE4A4 aggregate formation. Activation of autophagy with either thapsigargin or ionomycin inhibits PDE4A4 aggregate formation. Inhibition of autophagy with either wortmannin or LY294002 activates PDE4A4 aggregate formation. The protein kinase C inhibitors, RO 320432 and GO 6983, and the ERK inhibitors UO 126 and PD 98059 all activated PDE4A4 aggregate formation, whilst roscovitine, thalidomide and the tyrosine kinase inhibitors, genistein and AG17, all inhibited this process. We suggest that the fate of p62-containing protein aggregates need not necessarily be terminal, through delivery to autophagic vesicles and aggresomes. Instead, we propose a novel regulatory mechanism where a sub-population of p62-containing protein aggregates would form in a rapid, reversible manner so as to sequester specific cargo away from their normal, functionally important site(s) within the cell. Thus an appropriate conformational change in the target protein would confer reversible recruitment into a sub-population of p62-containing protein aggregates and so provide a regulatory function by removing these cargo proteins from their functionally important site(s) in a cell.