Glibenclamide quantification after intracranioventricular drug delivery.

<p>(<b>A</b>) Representative photograph of Nissl stained coronal section from a chronically ICV implanted rat (Scale bar: 500μm). (<b>B</b>) Glibenclamide concentrations in plasma (black circles) and CSF (grey circles) of rats given vehicle (n = 4) or glibenclamide (n = 6) by acute ICV injection. (<b>C</b>) CSF-to-plasma glibenclamide concentration ratio of rats given glibenclamide by acute ICV injection. All data are measurements from individual rats.</p

Plasma glibenclamide concentrations.

<p>(<b>A</b>) Glibenclamide concentration in plasma from individual female (white bars) and male (grey bars) mice implanted with a 21-day slow release 2.5mg glibenclamide pellet or a vehicle pellet. Data are mean±SEM of triplicate measurements. (<b>B</b>) Mean±SEM determined glibenclamide concentration in plasma from mice treated with a vehicle (n = 9 animals) or a 21-day slow release 2.5mg glibenclamide (n = 11 animals) pellet. (<b>C</b>) Mean±SEM determined glibenclamide concentration in plasma from female (white bars) and male (grey bars) mice implanted with a vehicle (n = 4 and n = 5 animals, respectively) or a 21-day slow release 2.5mg glibenclamide (n = 4 and n = 7 animals, respectively) pellet. ***<i>P</i><0.0001 [Two-way ANOVA followed by Bonferroni multiple comparison post-test].</p

Effect of subcutaneous glibenclamide therapy on blood glucose and isoflurane anaesthesia.

<p>(<b>A</b>) Free-fed blood glucose concentration of nV59M mice (black bars; n = 24) and control littermates (white bars; WT, n = 7; Nes-Cre⁺, n = 5; ROSA-V59M^+/-, n = 10) before and after implanting with a vehicle or 2.5mg glibenclamide slow-release subcutaneous pellet. The mean blood glucose of each mouse was averaged over a period of 5 days before and up to 7 days after pellet implantation. (<b>B,C</b>) Time taken for loss of righting (<b>B</b>) and withdrawal (<b>C</b>) reflexes in response to 2% isoflurane anaesthesia before (white bars) and one week after implanting nV59M mice (n = 24) and control littermates (WT, n = 7; Nes-Cre⁺, n = 5; ROSA-V59M^+/-, n = 10) with either a vehicle (grey bars) or 2.5mg glibenclamide (black bars) subcutaneous pellet. Half of the mice from each group were implanted with a vehicle pellet and the other half with glibenclamide. All data are mean±SEM. * P<0.05; n.s. not statistically significant. [Two-way ANOVA (genotype x treatment) followed by Bonferroni multiple comparison post-test].</p

Effect of intracranioventricular glibenclamide on the sensitivity to isoflurane.

<p>(<b>A</b>) Time taken for the LORR before (white bars) and one week after ICV infusion of vehicle (grey bars) or glibenclamide (138μg/ml, black bars) in nV59M (n = 12) and control (n = 27) mice. (<b>B</b>) Time taken for the LOWR before (white bars) and one week after ICV infusion of vehicle (grey bars) or glibenclamide (138μg/ml, black bars) in nV59M (n = 12) and control (n = 27) mice. (<b>C</b>) Representative Nissl-stained coronal section of mouse brain showing the site of the ICV cannula. (Scale bar: 500μm). Data are mean±SEM. n.s. not statistically significant [Two-way ANOVA (genotype x treatment) with <i>post-hoc</i> Bonferroni test].</p

Mass spectra of glibenclamide and d₁₁-glibenclamide.

<p>(<b>A,B</b>) Mass spectra and structural formulae of glibenclamide (<b>A</b>) and d₁₁-glibenclamide (<b>B</b>) in positive ionization mode obtained by direct injection ion trap mass spectrometry. Protonated molecular ions [M+H⁺] m/z (<b>A</b>) 494.1, (<b>B</b>) 505.1. Fragment ion m/z, (<b>A,B</b>) 369.0.</p

Effect of P-glycoprotein inhibition on glibenclamide concentrations.

<p>(<b>A</b>) Individual glibenclamide concentrations in plasma of rats injected intraperitoneally with 50mg/kg glibenclamide in the presence of elacridar (n = 11; grey bars) or vehicle (n = 10; black bars). (<b>B</b>) Individual glibenclamide concentrations measured in the CSF and brain homogenate of the rats injected with glibenclamide in the presence of elacridar (grey circles; n = 10 for CSF, n = 6 for brain) or vehicle (black circles; n = 9 for CSF, n = 6 for brain). (<b>C</b>) CSF-to-plasma and brain-to-plasma glibenclamide concentration ratio of rats injected IP with glibenclamide in the presence of elacridar (grey circles) or vehicle (black circles). Data are measurements from individual rats. The mean of each group is represented by the black bar. n.s. not statistically significant [Two-tailed unpaired Student’s t-test].</p

Effect of pellet drug dose on plasma glibenclamide concentration.

<p><b>(A)</b> Glibenclamide concentration in plasma from individual male mice implanted with a vehicle (n = 15 animals), a 0.25mg glibenclamide (n = 5 animals), a 2.5mg glibenclamide (n = 7 animals) or a 25mg glibenclamide pellet (n = 5 animals). <b>(B)</b> Glibenclamide concentration in plasma from individual male rats implanted with a vehicle (n = 10 animals), a 25mg glibenclamide (n = 5 animals) or a 200mg glibenclamide pellet (n = 5 animals). Data are measurements from individual animals. The mean of each group is represented by the black bar.</p

Sensitivity to isoflurane and halothane anaesthesia of nV59M mice.

<p>Time taken for loss of righting (<b>A</b>) and withdrawal (<b>B</b>) reflexes in response to 2% isoflurane or 2% halothane in 11–14 week-old nV59M mice (white bars; isoflurane: n = 41; halothane: n = 9) and control littermates (black bars; isoflurane: Nes-Cre⁺ n = 18, WT n = 15, ROSA-V59M^+/- n = 27; halothane: Nes-Cre⁺ n = 5; WT n = 2; ROSA-V59M^+/- n = 7). All data are mean ±SEM. ** <i>P</i><0.001; *** <i>P</i><0.0001 [Isoflurane: Mann-Whitney test; halothane: two-tailed unpaired t-test with Welch’s correction].</p

Glibenclamide quantification after intracranioventricular drug delivery.

<p>(<b>A</b>) Representative photograph of Nissl stained coronal section from a chronically ICV implanted rat (Scale bar: 500μm). (<b>B</b>) Glibenclamide concentrations in plasma (black circles) and CSF (grey circles) of rats given vehicle (n = 4) or glibenclamide (n = 6) by acute ICV injection. (<b>C</b>) CSF-to-plasma glibenclamide concentration ratio of rats given glibenclamide by acute ICV injection. All data are measurements from individual rats.</p

Effect of BSA on glibenclamide stimulated insulin release.

<p>Insulin secretion was evaluated in islets from 8–10 week-old male mice in response to 60–120 minute stimulation with various glucose and glibenclamide concentrations, as indicated, in the presence of (A) 15μmol/l BSA or (B) 0.9mmol/l BSA (n = 3 animals, 3 technical replicates per condition). Insulin secretion is expressed as a percentage of the insulin content. Insulin secretion in the presence of glucose and glibenclamide was significantly affected by the BSA concentration (<i>F(5</i>,<i>12)</i> = 10.77; <i>p</i> = 0.004). For both incubation times and for all drug concentrations tested, insulin secretion was significantly lower in the presence of 0.9mmol/l BSA than 15μM BSA (<i>p</i><0.05).</p