Cholinergic neural activity directs retinal layer-specific angiogenesis and blood retinal barrier formation.

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.

PDGFRß(+) cells in human and experimental neuro-vascular dysplasia and seizures.

INTRODUCTION: Neuro-vascular rearrangement occurs in brain disorders, including epilepsy. Platelet-derived growth factor receptor beta (PDGFRß) is used as a marker of perivascular pericytes. Whether PDGFRß(+) cell reorganization occurs in regions of neuro-vascular dysplasia associated with seizures is unknown. METHODS: We used brain specimens derived from epileptic subjects affected by intractable seizures associated with focal cortical dysplasia (FCD) or temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Tissues from cryptogenic epilepsy, non-sclerotic hippocampi or peritumoral were used for comparison. An in vivo rat model of neuro-vascular dysplasia was obtained by pre-natal exposure to methyl-axozy methanoic acid (MAM). Status epilepticus (SE) was induced in adult MAM rats by intraperitoneal pilocarpine. MAM tissues were also used to establish organotypic hippocampal cultures (OHC) to further assess pericytes positioning at the dysplastic microvasculature. PDGFRß and its colocalization with RECA-1 or CD34 were used to segregate perivascular pericytes. PDGFRß and NG2 or IBA1 colocalization were performed. Rat cortices and hippocampi were used for PDGFRß western blot analysis. RESULTS: Human FCD displayed the highest perivascular PDGFRß immunoreactivity, indicating pericytes, and presence of ramified PDGFRß(+) cells in the parenchyma and proximal to microvessels. Tissues deriving from human cryptogenic epilepsy displayed a similar pattern of immunoreactivity, although to a lesser extent compared to FCD. In TLE-HS, CD34 vascular proliferation was paralleled by increased perivascular PDGFRß(+) pericytes, as compared to non-HS. Parenchymal PDGFRß immunoreactivity co-localized with NG2 but was distinct from IBA1(+) microglia. In MAM rats, we found pericyte-vascular changes in regions characterized by neuronal heterotopias. PDGFRß immunoreactivity was differentially distributed in the heterotopic and adjacent normal CA1 region. The use of MAM OHC revealed microvascular-pericyte dysplasia at the capillary tree lining the dentate gyrus (DG) molecular layer as compared to control OHC. Severe SE induced PDGFRß(+) immunoreactivity mostly in the CA1 region of MAM rats. CONCLUSION: Our descriptive study points to microvascular-pericyte changes in the epileptic pathology. The possible link between PDGFRß(+) cells, neuro-vascular dysplasia and remodeling during seizures is discussed.

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.

In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags full-length endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of protein's distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.

In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin.

Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pH(p)) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pH(p) changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H(+) (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H(+) permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.

Discrepancy between CYP2D6 phenotype and genotype derived from post-mortem dextromethorphan blood level.

OBJECTIVE: To describe the death of a toddler after a therapeutic dose of dextromethorphan and its investigation. STUDY DESIGN: Case report, cytochrome P450 phenotype and genotype determination in the victim and post-mortem drug redistribution study performed in rats. RESULTS: A 20-month Asian male who received 3 mg of dextromethorphan once at 09:00 h and again at 22:00 h was found dead at 04:35 h. Post-mortem examination showed signs of early bronchopneumonia (bacterial cultures were negative); dextromethorphan and dextrorphan blood concentrations taken from the heart cavity were 500 ng/ml (1. 84 micromol/l) and 200 ng/ml (0.78 micromol/l), respectively. Despite the dextromethorphan level being almost 100-fold higher than expected after therapeutic doses, intentional or unintentional overdose was extremely unlikely; other potential causes were investigated. Post-mortem drug redistribution study performed in rats showed that dextromethorphan does not undergo extensive redistribution after death (6+/-5-fold increase) and could not explain the observed dextromethorphan level. The dextromethorphan/dextrorphan concentration ratio of 2.5 found in this toddler was compatible with a slow CYP2D6 metabolizer phenotype. However, the toddler exhibited a fast metabolizer genotype. Potential reasons for this discrepancy are discussed. CONCLUSION: CYP450 phenotypes derived from post-mortem blood levels should be interpreted with caution and preferably confirmed by a genotype analysis.

Noninvasive measurement of the pH of the endoplasmic reticulum at rest and during calcium release.

The pH within individual organelles of the secretory pathway is believed to be an important determinant of their biosynthetic activity. However, little is known about the determinants and regulation of the pH in the secretory organelles, which cannot be readily accessed by [H+]-sensitive probes. We devised a procedure for the dynamic, noninvasive measurement of pH in the lumen of the endoplasmic reticulum in intact mammalian cells. A recombinant form of the B subunit of Shiga toxin, previously modified to include a carboxyl-terminal KDEL sequence and a pH-sensitive fluorophore, was used for a two-stage delivery strategy. Retrograde traffic of endogenous lipids was harnessed to target this protein to the Golgi complex, followed by retrieval to the endoplasmic reticulum (ER) by KDEL receptors. Immunofluorescence and immunoelectron microscopy were used to verify the subcellular localization of the modified B fragment. Fluorescence ratio imaging and two independent calibration procedures were applied to determine the pH of the ER in situ. We found that the pH of the endoplasmic reticulum is near neutral and is unaffected during agonist-induced release of calcium. The ER was found to be highly permeable to H+ (equivalents), so that the prevailing [H+] is susceptible to alterations in the cytosolic pH. Plasmalemmal acid-base transporters were shown to indirectly regulate the endoplasmic reticulum pH.

The efficacy of diazepam in the treatment of acute iron overload in rats.

While conducting studies on the prevention of mortality from acute iron intoxication in rats, diazepam, given to prevent animal suffering, was observed to be associated with reduced mortality in a limited number of animals. The objective was to assess whether diazepam reduces mortality following acute iron intoxication in rats. Survival of rats was compared among groups receiving (i) orally 612 mg/kg iron alone (LD60), (ii) iron with a subcutaneous injection of 2.5 mg/kg diazepam (DZ), or (iii) iron, DZ with 800 mg/kg deferiprone intraperitoneal injections. The administration of DZ decreased mortality from 60 to 16% (p < 0.001). The addition of deferiprone to DZ resulted in zero mortality (p < 0.05 compared with the DZ group) over the study period. The administration of DZ was not associated with decreased iron absorption or increased urinary iron excretion, whereas the administration of deferiprone did result in urinary iron excretion. Microscopic examination suggests that diazepam administration may be associated with lower intracellular accumulation of iron. In conclusion, diazepam reduces mortality from iron overdose in rats through a yet unidentified mechanism, although the drug does not inhibit iron absorption or enhance urinary iron removal.

Interactions of clobazam with conventional antiepileptics in children.

Clobazam is a 1,5-benzodiazepine effective in antiepileptic therapy of children and adults. Presently it is mainly used as adjuvant therapy for intractable seizures. Our objective was to evaluate the effect of clobazam on the apparent clearance of other antiepileptic drugs at steady state, and to determine the factors that determine the plasma levels of clobazam and its active metabolite N-desmethylclobazam. Patients were 74 children with intractable seizures who received treatment with clobazam at our institution as part of the Canadian Cooperative Clobazam Study Group during the years 1987 to 1991. Serum concentrations of clobazam, N-desmethylclobazam, and of concomitant antiepileptic drugs were monitored and prospectively collected. The effect of clobazam treatment on the apparent clearance steady state of the other antiepileptic drugs was determined by statistical comparison of the clearances of each drug before and after initiation of clobazam treatment using Wilcoxon's signed rank test. The effects of dosage, age, and concomitant antiepileptic therapy on the levels of clobazam and N-desmethylclobazam was assessed by multivariate analysis. Response to treatment and incidence of adverse effects were evaluated for each conventional antiepileptic drug to possibly identify favorable or unfavorable combinations with clobazam. Whereas the clearances of most conventional antiepileptics are not affected by cotherapy with clobazam, the apparent clearances of valproic acid and primidone are significantly reduced in the presence of clobazam. Serum concentrations of clobazam increased with dosage and age, and decreased with phenobarbital cotherapy. Serum concentrations of N-desmethylclobazam significantly correlated with clobazam serum levels, age, or clobazam dosage and were significantly increased by cotherapy with phenytoin or carbamazepine. None of the concomitantly used drugs were associated with increased or decreased rate of seizure control. Twelve patients experienced mild adverse drug effects that were not associated with particular cotherapy, clobazam dose, or plasma concentrations. When clobazam is added to a therapy regimen that includes valproic acid, the patient should be closely followed for possible adverse drug reactions caused by elevated valproic acid serum concentrations, and monitoring of valproate serum levels should be considered. When clobazam doses are gradually increased to achieve an optimal clinical effect, the interactions with phenobarbital, carbamazepine, and phenytoin do not necessitate therapeutic drug monitoring of clobazam or N-desmethylclobazam, because there is a large therapeutic window and a poor correlation between plasma concentrations and therapeutic efficacy.

Efficacy of deferiprone in the treatment of acute iron intoxication in rats.

BACKGROUND: Deferiprone [(1,2-dimethyl-3-hydroxypyrid-4-one) (L1)], is the first orally active iron chelating agent to reach clinical trials in patients with chronic iron overload. Its efficacy in preventing morbidity and mortality in acute iron poisoning has not been tested. OBJECTIVE: To determine whether deferiprone can reduce the mortality of rats following toxic oral doses of iron. METHODS: Rats were administered 612 mg/kg elemental iron by gavage, corresponding to the LD58. A parallel group received the same oral dose of iron followed by deferiprone intraperitoneally at 400 mg/kg (loading dose), followed by additional intraperitoneal injections of 200 mg/kg, 100 mg/kg and 100 mg/kg of deferiprone at one hour intervals. RESULTS: Coadministering deferiprone with the iron decreased mortality from 58% (11/19) to 15% (3/20) (p = 0.013). The administration of deferiprone was associated with urinary excretion of iron (which did not occur with iron alone) and the production of the red deferiprone-iron complex. On histological examination there appeared to be less iron in the liver and gastrointestinal tract. CONCLUSION: The coadministration of deferiprone can decrease morbidity and mortality caused by acute iron overdose. Deferiprone holds promise for the treatment of iron poisoning but additional study is required.