ABSTRACT
We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins, and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV) or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1, and Kir3.2) and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miRNA). We show that AAV assembled to express HCN1 miRNA produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miRNA with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.
ABSTRACT
The present study tested the hypothesis that adenosine is involved in mediating the hyperemic response of the newborn brain to hypoglycemia. By use of the cranial window and microdialysis-H2 clearance methodologies, changes in the diameter of pial arterioles (25-50 microns), extracellular adenosine concentrations ([ADO]), and local cerebral blood flow (CBF) were examined in isoflurane-anesthetized piglets subjected to insulin-induced hypoglycemia. Blood glucose concentrations ranged from 10 to 18 mg/dl after insulin administration (25 IU/kg iv). Local CBF in the frontal cortex increased 36 +/- 12% (P = 0.014) at 30 min of hypoglycemia (group 1, n = 12; control = 43 +/- 3 ml.min-1.100 g-1). The mean increase in dialysate [ADO] sampled concurrently from the same cortical area was 59 +/- 29% (P = 0.011; control = 0.11 +/- 0.02 microM). At 30 min of hypoglycemia, pial diameters increased 55 +/- 10% (P = 0.001; group 2, n = 9). The [ADO] in cranial window cerebrospinal fluid (CSF) increased 217 +/- 71% (P = 0.04) in response to hypoglycemia (group 3, n = 8; control = 0.016 +/- 0.006 microM). Local administration of an adenosine antagonist, 10 microM 8-sulfophenyltheophylline, to the cerebral cortex before hypoglycemia caused a 38% reduction (P = 0.011) in the pial arteriolar response at 30 min of hypoglycemia (group 4, n = 9). Similarly, local superfusion of CSF with 3.7 mM glucose attenuated the hypoglycemia-induced pial dilation 33% (P = 0.039; group 5, n = 9). Perfusion of microdialysis probes with 3.7 mM glucose in the CSF abolished the hypoglycemia-induced increase in dialysate [ADO] (group 1).(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Adenosine/metabolism , Brain/metabolism , Cerebrovascular Circulation , Hyperemia/physiopathology , Hypoglycemia/physiopathology , Insulin/pharmacology , Adenosine/cerebrospinal fluid , Animals , Animals, Newborn , Arterioles/drug effects , Arterioles/physiology , Arterioles/physiopathology , Blood Glucose/metabolism , Blood Pressure , Carbon Dioxide/blood , Cerebrovascular Circulation/drug effects , Hyperemia/cerebrospinal fluid , Hyperemia/etiology , Hypoglycemia/cerebrospinal fluid , Hypoglycemia/chemically induced , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Muscle, Smooth, Vascular/physiopathology , Oxygen/blood , Partial Pressure , Pia Mater/blood supply , Regional Blood Flow , Swine , Theophylline/analogs & derivatives , Theophylline/pharmacology , Vasodilation/drug effectsABSTRACT
Serum creatine kinase BB-isoenzyme (CK-BB) activity was studied on the first day of life in 31 acutely asphyxiated infants, 70 infants born after high risk pregnancies (pre-eclampsia or intrauterine growth retardation, or both), and 47 very low birthweight infants. Neuro-developmental evaluation was carried out at 2.2-2.5 years. Eight infants died with, and eight without, hypoxic-ischaemic lesions of the brain, 14 had cerebral palsy, 16 had mild motor impairment, six had developmental delay without motor impairment, and 96 were normal at follow up. Infants who died with brain injury had significantly higher CK-BB activity than infants with normal outcomes (geometric mean 12 U/l); the mean difference was 82 U/l with a 95% confidence interval from 31 to 219 U/l. CK-BB in infants with cerebral palsy and mild motor impairment (geometric means 12 and 15 U/l, respectively) were similar to controls. CK-BB activity after birth is predictive of neonatal death but not of neurological damage in survivors.
Subject(s)
Brain Injuries/diagnosis , Clinical Enzyme Tests , Creatine Kinase/blood , Asphyxia Neonatorum/enzymology , Brain Injuries/mortality , Female , Fetal Growth Retardation/enzymology , Humans , Infant, Low Birth Weight , Infant, Newborn , Isoenzymes , Male , Pre-Eclampsia , Pregnancy , Prognosis , Risk FactorsABSTRACT
To assess the predictive value for perinatal brain damage of acidosis at birth, alone or in combination with the Apgar score at 5 minutes, a cohort of 982 liveborn infants delivered over two months was studied prospectively. The umbilical cord was double clamped, and arterial acid-base values were successfully determined in 964 infants and lactate concentration in 931. Reference values defining acidosis (mean +/- 2 SD) were obtained from a subset of 127 term infants who had no complications. The incidence of a low pH was 12% (111 out of 964), high base deficit 7% (70 out of 964), high lactate concentration 9% (83 out of 931), and low Apgar score at 5 minutes (less than or equal to 7) 3% (32 out of 982). Twelve of the 111 infants (11%) with acidosis had a low Apgar score, and 12 out of 29 infants (41%) with low Apgar scores had acidosis. At one year of age 35 infants were lost to follow up and 22 had an adverse outcome unrelated to asphyxia; 883 infants showed normal development but the possible sequelae of asphyxia were four deaths, slight abnormalities in 28 infants, and clear abnormalities in 10. The sensitivity and the positive predictive value of low pH for adverse outcome were, respectively, 21 and 8%, of high lactate concentration 12 and 5%, and of low 5 minute Apgar score 12 and 19%. Metabolic acidosis determined in blood from the umbilical artery at birth is a poor predictor of perinatal brain damage.