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A glucagon-like peptide-1 receptor agonist reduces intracranial pressure in a rat model of hydrocephalus

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Science Translational Medicine  23 Aug 2017:
Vol. 9, Issue 404, eaan0972
DOI: 10.1126/scitranslmed.aan0972
  • Fig. 1. GLP-1R expression in postmortem human choroid plexus tissue in vitro.

    (A) Representative image of hematoxylin and eosin staining of human choroid plexus tissue section demonstrating classic choroid plexus morphology. BV, blood vessel. (B) The histogram shows GLP-1R mRNA expression in human pancreas (n = 1), heart (n = 1), ovary (n = 1), and choroid plexus (n = 5). AU, arbitrary units. (C to D) Representative images of GLP-1R staining of paraffin-embedded human choroid plexus counterstained with hematoxylin. Sections were incubated without primary antibody (C) and with the human GLP-1R antibody MAb 3F52 (D). (E and F) High magnification of the boxed regions shown in (C) and (D), respectively. Scale bars, 100 μm.

  • Fig. 2. Expression of GLP-1R after treatment with exendin-4 in rat choroid plexus in vitro.

    (A) Representative images of rat choroid plexus after treatment with artificial CSF (aCSF) as control or fluorescently labeled exendin-4 (FLEX) in the presence or absence of the GLP-1R antagonist exendin 9-39. DAPI (4′,6-diamidino-2-phenylindole) (blue) was used as a nuclear marker. Scale bars, 50 and 25 μm (inset). (B to E) The histograms represent the fold change in mRNA expression of Glp-1r (B), Na+ K+ atpase (C), Aqp1 (D), and Nhe1 (E) (aCSF, n = 6; 3 hours, n = 7; 6 hours, n = 7). *P < 0.05, **P < 0.01; analysis of variance (ANOVA) with Tukey’s multiple comparisons test.

  • Fig. 3. Effect of exendin-4 treatment on cAMP and Na+ K+ ATPase activity in CPe cells.

    (A and B) The histograms represent the amount of cAMP generated after incubation with control, exendin-4 (ex-4) with and without 1 μM exendin 9-39 (ex 9-39), and forskolin (positive control) using two different methods of cAMP detection (A: control, n = 8; exendin-4, n = 8; forskolin, n = 5; B: control, n = 5; 1 nM exendin-4, n = 5; 10 nM exendin-4, n = 6; 100 nM exendin-4, n = 5; with 1 μM exendin 9-39, n = 6, 5, and 5, respectively). (C) Na+ K+ ATPase activity was measured by determining the concentration of inorganic phosphate generated by the hydrolysis of adenosine triphosphate that was sensitive to ouabain (Na+ K+ ATPase inhibitor) (control, n = 13; exendin-4, n = 7; PKI, n = 8; exendin-4 + PKI, n = 8). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant. Kruskal-Wallis test was followed by Mann-Whitney test (Bonferroni correction) (A) and ANOVA with Tukey’s multiple comparisons test (B and C).

  • Fig. 4. Effect of exendin-4 on ICP in healthy conscious rats.

    (A) Overview of the experimental design in normal rats. Rats were fitted with an epidural ICP probe and allowed to recover. Treatment was given daily for 5 days, and ICP was recorded on days 2, 4, and 6, before and after the rats received a subcutaneous (SC) injection of either saline (n = 9) or exendin-4 (20 μg/kg) (n = 9). (B) Example ICP traces of saline (blue) and exendin-4 (red) treatment. Spikes in the trace represent when the animal was moving (*), and accurate recording of ICP was confirmed by the response to jugular vein compression. (C to E) Line graphs showing the percentage of baseline ICP after subcutaneous injection of either saline or exendin-4 on day 2 (C), day 4 (D), and day 6 (E). (F and G) Histograms showing the pre-dose and 60-min posttreatment ICP values (% of baseline on day 2) on days 2, 4, and 6 for exendin-4 (F) and saline (G). (H) Line graph of the % change in weight from day 2 (start of treatment) showing that both saline- and exendin-4–treated rats lost weight but there was no significant difference between the groups on day 4 or 6. (I) Scatterplot of weight change (g) versus ICP change (mmHg) in the saline and exendin-4 groups. (J to N) Histograms showing blood pH (J) and CSF pH (K) and the concentrations of Na+ (L), Cl (M), and Ca2+ (N) in the CSF, 60 min after a subcutaneous injection of either saline or exendin-4 (20 μg/kg). (O) ICP was measured before and after an intracerebroventricular (ICV) injection of either 1-μl of saline (n = 8) or 0.3 μg of exendin-4 (n = 6). (P) Exendin 9-39 was continually infused (4 μg/μl per hour) into the lateral ventricle (ICV), and ICP was measured before and after a subcutaneous injection of either exendin-4 (20 μg/kg) (ICV exendin 9-39 + SC exendin-4, n = 6) or saline (ICV exendin 9-39 + SC saline, n = 5) and compared to continuous saline infusion (ICV saline + SC exendin-4, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Sidak’s multiple comparison test (C to H, O, and P) and t test (two-tailed) (J to N).

  • Fig. 5. Effects of different doses of exendin-4 on ICP, mRNA, and protein expression in healthy conscious rats.

    (A and B) Dose response of exendin-4’s effects on ICP after subcutaneous administration of exendin-4 (1 μg/kg, n = 6; 3 μg/kg, n = 6; 5 μg/kg, n = 23; and 20 μg/kg, n = 9) compared to saline (n = 18) at 30 and 60 min. (C) Line graph showing the percentage of baseline ICP after treatment with exendin-4 (1, 3, or 5 μg/kg) measured over 3 hours. (D to G) The histograms show Glp-1r (D), Na+ K+ atpase (E), Aqp1 (F), and Nhe1 (G) mRNA expression in the rat choroid plexus after saline treatment (n = 4) or treatment with exendin-4 (1 μg/kg, n = 5; 3 μg/kg, n = 6; and 5 μg/kg, n = 6). (H) Representative Western blots and (I to K) semiquantitative protein analysis for (I) Na+ K+ ATPase (112 kDa) and (J) total AQP1, either nonglycosylated (NG) (29 kDa) or glycosylated (G) (35 kDa); β-actin (42 kDa) was used as loading control. (K) Histogram shows the ratio of glycosylated to nonglycosylated AQP1. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons test (B and C) and ANOVA with Tukey’s multiple comparisons test (D to G and I to K).

  • Fig. 6. Effects of exendin-4 time course on ICP, mRNA, and protein expression in healthy conscious rats.

    (A) Line graph showing the percentage of baseline ICP after a single subcutaneous injection of saline (n = 18) or exendin-4 (5 μg/kg) (n = 24) measured over 24 hours. (B to D) Histograms showing weight loss (B), water intake (C), and food intake (D) in rats treated with saline (n = 4) or exendin-4 (5 μg/kg) at 3 hours (n = 6), 6 hours (n = 6), and 24 hours (n = 6). (E to H) Histograms representing Glp-1r (E), Na+ K+ atpase (F), Aqp1 (G), and Nhe1 (H) mRNA expression in the rat choroid plexus after treatment with saline (n = 4) and exendin-4 (5 μg/kg) at 3 hours (n = 6), 6 hours (n = 5), and 24 hours (n = 5). (I) Representative Western blots and (J to L) semiquantitative protein analysis for (J) Na+ K+ ATPase (112 kDa) and (K) total AQP1, either nonglycosylated (29 kDa) or glycosylated (35 kDa). β-actin (42 kDa) was used as loading control. (L) The histogram shows the ratio of glycosylated to nonglycosylated AQP1. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; two-way ANOVA with Sidak’s multiple comparisons test (A to D) and ANOVA with Tukey’s multiple comparisons test (E to H and J to L).

  • Fig. 7. Effect of exendin-4 on ICP in a rat model of raised ICP (hydrocephalic).

    (A) Overview of the experimental plan. Kaolin was injected into the cisterna magna to induce hydrocephalus. On day 6, the ICP monitor was implanted under anesthesia, and ICP was recorded overnight to allow the ICP to normalize after implantation. On day 7, the rats were given a subcutaneous injection of either saline (n = 6) or exendin-4 (20 μg/kg) (n = 6), and the ICP was recorded for a further 60 min. (B) Dot plot showing the individual baseline ICP values (mmHg) for the normal rats and rats injected with kaolin. The kaolin group had significantly higher baseline ICP values compared to the normal group, with 8 of 12 rats having an ICP value of >10 mmHg. (C) Line graph showing the percentage of baseline ICP after treatment with either saline (light blue; n = 6) or exendin-4 (light red; n = 6). The groups could also be further divided into those with an ICP value of >10 mmHg in the saline group (dark blue; n = 4) and exendin-4 group (dark red; n = 4). (D) Example ICP trace in a hydrocephalic rat before and after treatment with exendin-4. Before treatment, the rat exhibited pathological ICP B-waves (b), which were abolished after treatment with exendin-4. ****P < 0.0001, t test (two-tailed) (B) and two-way ANOVA with Sidak’s multiple comparisons test (C).

Supplementary Materials

  • Supplementary Material for:

    A glucagon-like peptide-1 receptor agonist reduces intracranial pressure in a rat model of hydrocephalus

    Hannah F. Botfield, Maria S. Uldall, Connar S. J. Westgate, James L. Mitchell, Snorre M. Hagen, Ana Maria Gonzalez, David J. Hodson, Rigmor H. Jensen, Alexandra J. Sinclair*

    *Corresponding author. Email: a.b.sinclair{at}bham.ac.uk

    Published 23 August 2017, Sci. Transl. Med. 9, eaan0972 (2017)
    DOI: 10.1126/scitranslmed.aan0972

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Characterization of primary rat CPe cells in vivo and in vitro.
    • Fig. S2. Suggested route for GLP-1 action at the choroid plexus.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Individual level data corresponding to the different figures (provided as an Excel file).

    [Download Table S1]

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