Research ArticleHYDROCEPHALUS

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

Could a drug for diabetes help in the treatment of hydrocephalus?

Hydrocephalus is a life-threatening condition in babies caused by raised intracranial pressure due to an increase in cerebrospinal fluid volume. Now, Botfield et al. show that the glucagon-like peptide-1 receptor (GLP-1R), known to modulate fluid homeostasis in the kidney, is expressed in human and rodent choroid plexus, the brain area responsible for cerebrospinal fluid secretion. The authors show that treating a rat model of hydrocephalus with a GLP-1R agonist reduced intracranial pressure. This suggests that GLP-1R agonists, approved for treating diabetes, could be repurposed for treating hydrocephalus and potentially other conditions characterized by raised ICP.

Abstract

Current therapies for reducing raised intracranial pressure (ICP) under conditions such as idiopathic intracranial hypertension or hydrocephalus have limited efficacy and tolerability. Thus, there is a pressing need to identify alternative drugs. Glucagon-like peptide-1 receptor (GLP-1R) agonists are used to treat diabetes and promote weight loss but have also been shown to affect fluid homeostasis in the kidney. We investigated whether exendin-4, a GLP-1R agonist, is able to modulate cerebrospinal fluid (CSF) secretion at the choroid plexus and subsequently reduce ICP in rats. We used tissue sections and cell cultures to demonstrate expression of GLP-1R in the choroid plexus and its activation by exendin-4, an effect blocked by the GLP-1R antagonist exendin 9-39. Acute treatment with exendin-4 reduced Na+- and K+-dependent adenosine triphosphatase activity, a key regulator of CSF secretion, in cell cultures. Finally, we demonstrated that administration of exendin-4 to female rats with raised ICP (hydrocephalic) resulted in a GLP-1R–mediated reduction in ICP. These findings suggest that GLP-1R agonists can reduce ICP in rodents. Repurposing existing GLP-1R agonist drugs may be a useful therapeutic strategy for treating raised ICP.

INTRODUCTION

Elevated intracranial pressure (ICP) is caused by alterations in the volume of either cerebral blood, cerebrospinal fluid (CSF), or brain tissue. CSF volume is tightly regulated and depends on the balance between CSF secretion, which is modulated predominantly by the choroid plexus, and drainage through the arachnoid granulations and lymphatics (1). Reducing CSF volume, by either CSF drainage or decreasing CSF secretion, is used therapeutically to lower ICP (2, 3) under conditions characterized by raised ICP such as idiopathic intracranial hypertension and hydrocephalus.

In the choroid plexus, CSF is secreted by the choroid plexus epithelial (CPe) cells and is driven by net movement of sodium ions (Na+) from the blood into the cerebral ventricles. This creates an osmotic gradient, which drives water transport into the cerebral ventricles. There are numerous ion channels involved in this process, but the apical Na+- and K+-dependent adenosine triphosphatase (Na+ K+ ATPase) that pumps Na+ into the ventricles is the most important of these channels and represents the rate-limiting step (4, 5). Specific inhibition of the Na+ K+ ATPase with ouabain reduces CSF secretion by 70 to 80% (6). Hence, the CPe cells function akin to inverted renal proximal tubule epithelial cells with an analogous mechanism of fluid transport (7, 8).

The incretin glucagon-like peptide-1 (GLP-1) is a gut peptide secreted by the distal small intestine in response to food intake (9). GLP-1 stimulates glucose-dependent insulin secretion and inhibits glucagon release, lowering blood glucose (10). In addition, GLP-1 is synthesized in neurons of the nucleus tractus solitarius, which project to the hypothalamus (11) and promote satiety and weight loss (1214). GLP-1 signals through the GLP-1 receptor (GLP-1R), a class B G protein (heterotrimeric GTP-binding protein)–coupled receptor expressed in selected cell types within the central nervous system including the hypothalamus, hippocampus, olfactory cortex, circumventricular organs, hindbrain, and choroid plexus (1517).

GLP-1 also has effects on renal proximal tubule Na+ secretion, reducing Na+ reabsorption and increasing diuresis (18). Here, GLP-1R activation stimulates the conversion of adenosine triphosphate to cyclic adenosine monophosphate (cAMP) by adenylate cyclase. cAMP activates protein kinase A (PKA), which inhibits the Na+ H+ exchanger, thereby preventing Na+ reabsorption into the bloodstream (18). The diuretic actions of incretins have led to investigation of their use as antihypertensive agents (19). Similar to its activity in the kidney, we hypothesize that GLP-1 also modulates Na+ transport and, subsequently, fluid movement at the choroid plexus. We propose that GLP-1R activation may inhibit the basal Na+ H+ exchanger through cAMP-dependent PKA activation, thus impeding the Na+ K+ ATPase–dependent secretion of CSF. Stabilized GLP-1 mimetics are widely used to treat diabetes and obesity and therefore could be repurposed for treating raised ICP.

Here, we used tissue sections and CPe cell cultures to assess the localization and distribution of the GLP-1R in rat and human choroid plexus and determined the effects of GLP-1R stimulation on CSF secretion. Furthermore, we conducted in vivo studies to evaluate the effects of GLP-1R agonists on ICP in a hydrocephalus rat model with raised ICP.

RESULTS

The GLP-1R is expressed in human choroid plexus tissue

Immunohistochemical analysis using hematoxylin and eosin staining confirmed that human donor tissue comprised the choroid plexus, demonstrating choroid plexus morphology including a cuboidal CPe cell monolayer resting on a basement membrane, the underlying interstitial tissue, and capillary vessels (Fig. 1A). GLP-1R mRNA expression in five human choroid plexus samples was compared to known commercially available GLP-1R–positive tissues (pooled samples; see Materials and Methods for source details). Human pancreas had the highest expression of GLP-1R mRNA, with the heart and ovary having the least. Human choroid plexus showed GLP-1R mRNA expression (Fig. 1B). To determine the localization of the receptor protein, we immunostained paraffin-embedded human choroid plexus sections with a specific monoclonal antibody to human GLP-1R previously validated in human and monkey tissue sections (20, 21). On the basis of the morphology of the choroid plexus, GLP-1R–positive staining was detected in CPe cells (Fig. 1, C to F). Together, these studies demonstrate that the human choroid plexus expresses GLP-1R mRNA and protein.

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.

Exendin-4 treatment modulates the GLP-1R in rat choroid plexus in vitro

Given the lack of validated antibodies against rodent GLP-1R, we instead incubated whole rat choroid plexus in vitro with a fluorescently tagged GLP-1R agonist, exendin-4 (FLEX), to demonstrate the presence of the receptor in the choroid plexus. After 15 min of 1 μM FLEX incubation, only a few CPe cells were positive for FLEX within the cytoplasm (Fig. 2A). However, this increased by 30 min (Fig. 2A). In both cases, GLP-1R appeared to localize predominantly in the cytoplasm, consistent with agonist-induced receptor internalization and trafficking, most likely via endosomes (22). The GLP-1R antagonist exendin 9-39 (1 μM) reduced the number of FLEX-positive cells within the choroid plexus (Fig. 2A), suggesting specific binding of the FLEX ligand to GLP-1R.

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.

Next, we determined Glp-1r mRNA expression in whole rat choroid plexus tissue after incubation with 100 nM exendin-4. Incubation of the rat choroid plexus with exendin-4 for 3 hours showed an increase in Glp-1r mRNA compared to artificial CSF (3.21 ± 0.70–fold; P < 0.01), with a return to baseline at 6 hours (0.78 ± 0.12–fold) (Fig. 2B). There was also a small but detectable increase in Na+ K+ atpase mRNA expression after 3 hours of exendin-4 treatment compared to incubation with artificial CSF (1.82 ± 0.28–fold; P < 0.05), which again returned to baseline at 6 hours (0.97 ± 0.21–fold) (Fig. 2C). The expression of other channels and transporters involved in CSF secretion, including the water channel aquaporin 1 (Aqp1) and the Na+ H+ exchanger (Nhe1), was not altered after exendin-4 treatment (Fig. 2, D and E).

Exendin-4 treatment increases cAMP and reduces Na+ K+ ATPase activity

To explore further the effects of exendin-4 on the choroid plexus, we grew monolayers of rat CPe cells in culture. These CPe cells were characterized using antibodies against specific identity markers and were shown to be similar to their in vivo counterparts (fig. S1A), including the expression of Glp-1r mRNA (fig. S1B). To determine the effect of exendin-4 on GLP-1R signaling, we assessed cAMP generation using two enzyme immunoassay systems. Treatment of CPe cells with exendin-4 increased cAMP compared to control (2.14 ± 0.61–fold; P < 0.01) (Fig. 3A) in a concentration-dependent manner, and this could be inhibited by exendin 9-39 (Fig. 3B). Forskolin, an adenylate cyclase activator, was used as a positive control to maximally stimulate cAMP production (5.30 ± 0.74–fold compared to control) (Fig. 3, A and B).

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).

The role of GLP-1R signaling in CSF secretion was assessed in rat CPe cell cultures by measuring Na+ K+ ATPase activity (proposed as a marker of CSF secretion from the choroid plexus) (6). Exendin-4 treatment reduced Na+ K+ ATPase–specific phosphate production compared to control (39.3 ± 9.4%; P < 0.05) (Fig. 3C). In addition, inhibition of PKA with PKI(16–22) amide [PKA inhibitor (PKI)] abolished the exendin-4–induced reduction in Na+ K+ ATPase activity (95.4 ± 17.6%; P < 0.05) (Fig. 3C).

Exendin-4 treatment reduces ICP in conscious rats

To establish whether exendin-4 was able to modulate ICP in vivo, we implanted healthy female adult rats with an ICP monitor (day 0) before they received daily subcutaneous injections of either saline or exendin-4 (20 μg/kg) for 5 days (day 2 to 6). ICP was measured before and after the subcutaneous injection on days 2, 4, and 6 (Fig. 4A). Examples of the ICP traces are shown in Fig. 4B. On the first day of treatment (day 2), exendin-4 significantly reduced ICP 10 min after the subcutaneous injection; by 30 min, ICP was 65.2 ± 6.6% of baseline compared to 91.0 ± 3.9% of baseline in saline-treated rats (P < 0.01) (Fig. 4C). A similar drop in ICP was observed on day 4 (50.4 ± 6.9% of baseline; P < 0.001) and day 6 (54.5 ± 8.2% of baseline; P < 0.001) 30 min after exendin-4 administration (Fig. 4, D and E).

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).

In addition to reducing ICP immediately after treatment, exendin-4 had a cumulative effect on reducing ICP. Exendin-4 caused a significant reduction in ICP measured before dose on day 2 (baseline, 100%) to day 4 (79.3 ± 7.3%; P < 0.05) and day 6 (72.5 ± 5.6%; P < 0.01) (Fig. 4F), which was not observed in saline-treated rats [day 2, 100% (baseline); day 4, 95.5 ± 13.6%; day 6, 105.3 ± 12.5%; Fig. 4G].

Because there is evidence that weight loss can alter ICP (23), weights were monitored over the treatment period. Although both saline- and exendin-4–treated rats lost weight during treatment (P < 0.05), there was no significant difference between the groups at any time point (Fig. 4H). In the saline group, weight change correlated with alterations in ICP (r = 0.710, P = 0.032), although no relationship was detected for the exendin-4 treatment group (r = −0.300, P = 0.552) (Fig. 4I).

The effect of subcutaneous administration of exendin-4 (20 μg/kg) on blood and CSF pH and CSF electrolytes was analyzed 60 min after treatment. Exendin-4 maintained normal blood pH (7.35 ± 0.01; Fig. 4J); however, it caused a reduction in CSF pH (7.41 ± 0.03; P < 0.05) compared to saline (blood pH, 7.35 ± 0.03; CSF pH, 7.61 ± 0.07) (Fig. 4K). CSF Na+ concentration remained unaltered (saline, 150.3 ± 0.9 mM; exendin-4, 150.3 ± 0.6 mM) (Fig. 4L), whereas the concentration of Cl ions in the CSF was reduced in the exendin-4 group (117 ± 0.5 mM; P < 0.05) compared to the saline group (123.8 ± 0.9 mM) (Fig. 4M). Exendin-4 treatment also increased the concentration of Ca2+ ions in the CSF (1.09 ± 0.01 mM; P < 0.05) compared to saline (1.03 ± 0.02 mM) (Fig. 4N).

Exendin-4 acts via GLP-1R in the brain to reduce ICP in rats

To assess whether the reduction in ICP was specific to the brain, we injected exendin-4 into the lateral ventricle through an intracerebroventricular cannula in anesthetized rats. Intracerebroventricular delivery of exendin-4 reduced ICP over time, which was significantly different from baseline at 15 min (68.9 ± 6.4%; P < 0.05). Intracerebroventricular delivery of saline also reduced ICP over time (technical effect due to the intracerebroventricular cannula itself reducing ICP), and this was significantly different from baseline at 50 min (74.5 ± 7.9%; P < 0.05). Over the 60-min ICP measurement, intracerebroventricular delivery of exendin-4 significantly reduced the area under the curve (AUC) of ICP compared to saline delivered via the same route (3852 ± 397 versus 4974 ± 262 AUC; P < 0.05) (Fig. 4O). To determine whether the effects of exendin-4 on ICP were mediated by the GLP-1R, we continuously infused the antagonist exendin 9-39 (4 μg/hour) into the lateral ventricle for 2 days before subcutaneous administration of either saline or exendin-4 (20 μg/kg). Subcutaneous injection of exendin-4 (ICV saline + SC exendin-4) lowered ICP (P < 0.0001) compared to a subcutaneous injection of saline with intracerebroventricular delivery of exendin 9-39 (ICV exendin 9-39 + SC saline; Fig. 4P). Central intracerebroventricular exendin 9-39 infusion decreased the ICP-lowering effect of subcutaneous exendin-4 at 5 min (ICV exendin 9-39 + SC exendin-4, 96.7 ± 13.7% versus ICV saline + SC exendin-4, 65.7 ± 8.3%; P < 0.001) (Fig. 4P). These data suggest that exendin-4 in part exerts its effects on ICP via the GLP-1R signaling pathway in the brain.

Exendin-4 reduces ICP in a dose-dependent manner and the effects last for 24 hours

Rats were treated subcutaneously with exendin-4 (1, 3, and 5 μg/kg) to determine whether exendin-4 reduces ICP at lower concentrations. At 60 min, exendin-4 (1, 3, and 5 μg/kg) significantly reduced ICP to 79.0 ± 7.0% of baseline (P < 0.05), 69.9 ± 8.8% of baseline (P < 0.0001), and 60.6 ± 3.6% of baseline (P < 0.0001), respectively, compared to saline (97.2 ± 2.5% of baseline) (Fig. 5, A and B). Exendin-4 (5 μg/kg) showed the greatest reduction in ICP, and the effect was still present 3 hours after the treatment (P < 0.001). Conversely, in the 1 and 3 μg/kg exendin-4 groups, ICP had returned to baseline by 3 hours (Fig. 5C).

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).

Alterations in mRNA and protein expression of GLP-1R and molecules involved in CSF secretion were assessed in the choroid plexus of rats 3 hours after treatment with exendin-4 (1, 3, and 5 μg/kg). Glp-1r and Na+ K+ atpase mRNA expression was not altered by exendin-4 treatment (Fig. 5, D and E). Conversely, there was a twofold increase in the amount of Aqp1 mRNA in the 5 μg/kg exendin-4 treatment group (P < 0.05) (Fig. 5F) and a fourfold increase in the amount of Nhe1 mRNA expression in the 1 μg/kg exendin-4 treatment group (P < 0.05) (Fig. 5G). Although no significant changes were observed in Na+ K+ atpase mRNA expression, there was a small increase in Na+ K+ ATPase protein in the 5 μg/kg exendin-4 treatment group (2.16 ± 0.22 AU; P < 0.05) (Fig. 5, H and I). Two bands were observed for the water channel aquaporin 1 (AQP1), representing the glycosylated (top band) and nonglycosylated (bottom band) forms of AQP1 (Fig. 5H). The total amount of AQP1 protein was slightly reduced by the 1 and 3 μg/kg exendin-4 treatment but not with the higher 5 μg/kg exendin-4 dose (1 μg/kg, 1.96 ± 0.17 AU, P < 0.05; 3 μg/kg, 1.75 ± 0.08 AU, P < 0.01; 5 μg/kg, 2.75 ± 0.30 AU) (Fig. 5J). The ratio of glycosylated AQP1 to nonglycosylated AQP1 was increased after the 1 and 3 μg/kg exendin-4 treatment but not after the 5 μg/kg exendin-4 treatment (1 μg/kg, 0.97 ± 0.06 AU, P < 0.05; 3 μg/kg, 1.08 ± 0.06 AU, P < 0.01; 5 μg/kg, 0.81 ± 0.08 AU) (Fig. 5K). Glycosylation is important for intracellular trafficking and protein stability, making proteins more resistant to proteolysis (24); therefore, these data suggest that exendin-4 may lower AQP1 through enhanced degradation of the nonglycosylated AQP1.

The effect of exendin-4 (5 μg/kg) was monitored for 24 hours in healthy rats to determine its duration of action. A single subcutaneous injection of exendin-4 (5 μg/kg) maintained lower ICP compared to saline over 24 hours and returned to the pre-dose ICP baseline at 24 hours (1 hour, 60.2 ± 3.5%, P < 0.0001; 3 hours, 71.3 ± 3.7%, P < 0.001; 6 hours, 70.3 ± 4.0%, P < 0.0001; 12 hours, 88.9 ± 16.6%, P < 0.01; 24 hours, 100.3 ± 14.3%, P < 0.01) (Fig. 6A). Effects on weight and food and water intake were also noted in relation to changes in ICP over 24 hours. Although exendin-4 caused a greater reduction in weight at 3 and 6 hours (Fig. 6B), there were no differences between food or water intake at any time point between exendin-4–treated and saline-treated rats (Fig. 6, C and D). Glp-1r, Na+ K+ atpase, and Nhe1 mRNA expression did not change over the 24-hour period (Fig. 6, E, F, and H). As shown previously, exendin-4 (5 μg/kg) increased Aqp1 mRNA expression at 3 hours compared to saline, although this was not observed at any other time point (Fig. 6G). There were also no significant changes in the amount of Na+ K+ ATPase or AQP1 protein over the 24-hour time period (Fig. 6, I to L).

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).

Exendin-4 treatment reduces ICP in a rodent model of raised ICP

To determine the efficacy of exendin-4 to reduce ICP under conditions of raised ICP, we used a well-characterized kaolin model of hydrocephalus in rats. Kaolin, an aluminum silicate, acts as an irritant, inducing an inflammatory response with concomitant deposition of collagen and dense fibrosis in areas of the subarachnoid space close to the injection site, which leads to raised ICP (25, 26). Kaolin was injected into the cisterna magna, leading to development of hydrocephalus, before implantation of the ICP monitor. ICP was recorded before and after a subcutaneous injection of either saline or exendin-4 (20 μg/kg) (Fig. 7A). The injection of kaolin significantly increased baseline ICP (11.1 ± 1.3 mmHg; P < 0.0001) compared to that of normal rats (5.5 ± 0.4 mmHg) (Fig. 7B). Exendin-4 treatment significantly reduced ICP almost immediately after the subcutaneous injection and, at 30 min, was 62.6 ± 5.1% of baseline (P < 0.0001) compared to 105.0 ± 4.6% of baseline in saline-treated rats (Fig. 7C). Eight rats in the kaolin group had baseline ICP values of greater than 10 mmHg and had an average baseline ICP of 13.7 ± 0.7 mmHg. In these rats (ICP > 10 mmHg), the ICP values at 30 min were 56.6 ± 5.7% of baseline (n = 4) in the exendin-4 treatment group compared to 106.7 ± 8.6% of baseline (n = 4) in the saline treatment group (Fig. 7C). In the rodents with elevated ICP, the ICP waveform was very unstable, with the appearance of B-waves characteristic of pathologically elevated ICP and a reduction in brain compliance (27). These were abolished in rats receiving exendin-4 but not saline (Fig. 7D).

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).

DISCUSSION

The aim of the present study was to establish whether GLP-1 had a role in modulating CSF secretion and ICP. We were able to demonstrate that the GLP-1R agonist exendin-4 was able to reduce ICP in conscious healthy female rats and in a rat model of raised ICP. In addition, our results suggest that the ICP-lowering properties of exendin-4 may occur through reduced CSF secretion at the choroid plexus, implied by the reduction in Na+ K+ ATPase activity in CPe cells. Furthermore, our data suggest that exendin-4 modulates CSF production in vitro through the GLP-1R/cAMP/PKA signaling pathway.

Alvarez et al. (15) first described the presence of the GLP-1R in the rat ependyma and choroid plexus by in situ hybridization but did not characterize the cellular localization of this receptor. Our studies corroborate these findings and demonstrate further that GLP-1R mRNA and protein are present in both rat and human choroid plexus. We localized the GLP-1R protein in tissue sections of the human choroid plexus to the CPe cells using a monoclonal antibody and showed the presence of the receptor in the rat choroid plexus using fluorescently tagged exendin-4. We note that no specific antibody exists for mouse/rat tissue, so rodent tissue was not examined for GLP-1R protein expression. In any case, our studies are in keeping with others showing localization of the GLP-1R in monkey kidney and human GLP-1R transfected cells (20, 21). G protein–coupled receptors undergo internalization, trafficking, and recycling/degradation after agonist stimulation (28). We speculate that such dynamics may allow the GLP-1R to be stimulated from both sides of the choroid plexus (fig. S2A). Although GLP-1R mRNA and protein expression were, in general, low, it has recently been shown that activation of the receptor requires femto- to picomolar concentrations of GLP-1R, so even faced with low abundance, signaling would be expected in the presence of exendin-4 (29).

We successfully cultured monolayers of rat CPe cells, which we used as an in vitro cell culture model of the rat choroid plexus to assess CSF secretion. The Na+ K+ ATPase is localized to the apical surface and is the driving force for transporting Na+ ions from the choroid plexus into the CSF against its concentration gradient. Many studies have demonstrated that modulation of Na+ K+ ATPase expression or activity directly correlates with CSF secretion (6, 3033). We were able to show that exendin-4 reduces Na+ K+ ATPase activity, suggesting reduced CSF secretion at the choroid plexus. Previous studies have shown similar effects of exendin-4 on Na+ K+ ATPase activity in the renal system (34). In kidney proximal tubule epithelial cells and pancreatic β cells, GLP-1 modulates Na+ concentration through increased cAMP and PKA activation (18, 35). Using two different techniques, exendin-4 was seen to induce a concentration-dependent rise in cAMP in the choroid plexus, which was inhibited by the GLP-1R antagonist exendin 9-39. Furthermore, a PKI blocked the effects of exendin-4 on Na+ K+ ATPase activity, although we acknowledge that such approaches can be nonspecific and further studies using specific knockout animals are required. Altogether, these data indicate that the cAMP/PKA-dependent pathway may be involved in the GLP-1R–mediated reduction in CSF secretion at the choroid plexus. In the kidney, GLP-1R agonist treatment increases diuresis through phosphorylation of the Na+ H+ exchanger (18, 36). There are PKA phosphorylation sites present on both the Na+ H+ exchanger and the Na+ K+ ATPase (37); therefore, in the choroid plexus, phosphorylation of either the Na+ H+ exchanger or the Na+ K+ ATPase may result in inhibition of Na+ transport across the cells and thus CSF production (fig. S2, B and C). In the choroid plexus, the Na+ K+ ATPase can also be phosphorylated by PKC (37). GLP-1R is able to signal through the PKC pathway in pancreatic β cells (29, 38, 39). Therefore, the GLP-1R/PKC signaling pathway may also have a role in reducing CSF secretion and warrants further investigation.

The key finding of this study is that subcutaneous exendin-4 treatment is able to reduce ICP in vivo in normal rats and rats with raised ICP. In addition, the effect on ICP of a single administration of exendin-4 lasted for 24 hours, and cumulative dosing reduced the pre-dose ICP. This suggests that exendin-4 may be able to maintain low ICP over a long period. This is an important advance, because there are very limited specific therapeutic options to clinically reduce and maintain low ICP under conditions of raised ICP. The main therapeutic agent for managing chronic raised ICP is acetazolamide, a carbonic anhydrase inhibitor. However, in idiopathic intracranial hypertension, acetazolamide is associated with limited efficacy and poor tolerability (48% withdrawal) (2) and is contraindicated for use in premature infants with post-hemorrhagic hydrocephalus (40). On the other hand, treatment with incretin mimetics is generally well tolerated, with the main side effects being transient nausea, constipation, and diarrhea, and these drugs do not induce hypoglycemia (41). In patients taking the GLP-1R agonist liraglutide, drug withdrawal due to side effects was only 5.4% in the cohort receiving the highest dose [3 mg; (12)].

However, there are a number of limitations to the present study. To determine the central actions of exendin-4 on ICP, we had to deliver exendin-4 directly into the brain’s ventricular system. The injection itself may have a direct effect on ICP and could mask any changes in ICP relating to the treatment. To try to minimize these effects, we implanted an intracerebroventricular cannula 2 days before the injection. Nonetheless, because it was not possible to completely seal the system, ICP showed a slight decrease in saline-treated rats. However, we were still able to establish a significant reduction in ICP with exendin-4 treatment. The study design was also limited by the lack of blinding during the intervention, although the data were analyzed by different individuals with the same outcome. ICP was monitored continuously via automated software, thus removing measurement bias. It will be of interest to study prolonged dosing in a rat model of hydrocephalus in the future. However, this will require considerable technical optimization, given that ICP is notoriously difficult to measure in such models where recordings are typically only accurate immediately before euthanasia (42, 43).

GLP-1R agonists also have peripheral actions that have the potential to indirectly affect ICP. Although incretin mimetics have been shown to acutely increase heart rate and blood pressure (44), hypertension would be expected to cause the opposite effect to that seen here due to increased choroid plexus permeability and fluid secretion (45, 46). Our data imply that the effect of exendin-4 on ICP dynamics is through central mechanisms, because intracerebroventricular infusion of exendin 9-39 partially inhibited the action of subcutaneous exendin-4. Exendin 9-39 may not have fully inhibited the actions of exendin-4, because the inhibitor was infused into the ventricle rather than being given as a bolus injection. However, it is also possible that the effects of exendin-4 are not fully mediated by GLP-1R, and this requires further investigation. Previous studies have also demonstrated only moderate effects on attenuating exendin-4–induced food intake suppression at early time points after intracerebroventricular bolus of exendin 9-39 (47). Nevertheless, the central actions of exendin-4 are further supported by the fact that exendin-4 lowered CSF pH, whereas blood pH remained unchanged, which is supported by other studies showing that GLP-1 does not affect blood pH (19). It remains unclear how the subcutaneous administration of the GLP-1R agonist exendin-4 exerts its central effects on the choroid plexus. After subcutaneous administration, circulating exendin-4 may cross the fenestrated capillaries in the choroid plexus and stimulate the GLP-1R on the basolateral side of the CPe cells. Otherwise, it is possible that exendin-4 crossed the blood-brain barrier (48, 49) or entered the CSF via the circumventricular organs, where it is able to stimulate the receptors on the apical surface of the CPe cells. Liraglutide readily crosses into the hypothalamic arcuate nucleus (50), and in vivo imaging studies in rodents using fluorescently tagged ghrelin show passage of the gut peptide to the same region via fenestrated capillaries of the median eminence (51). Last, exendin-4 may stimulate vagal afferents that project to the nucleus tractus solitarius (11). This may lead to secretion of GLP-1 through a widespread network of fibers projecting to the third ventricle, allowing GLP-1 to enter the CSF (fig. S2A).

In summary, exendin-4 reduces Na+ K+ ATPase activity at the choroid plexus, implying a reduction in CSF secretion, and lowers ICP in conscious rats with and without elevated ICP. This work demonstrates that GLP-1R agonists may provide an alternative treatment for raised ICP under conditions such as idiopathic intracranial hypertension and hydrocephalus and warrant further clinical investigation in humans.

MATERIALS AND METHODS

Study design

The main aim of this study was to explore the potential of exendin-4, a GLP-1R agonist, to modulate CSF secretion and subsequently reduce ICP. Three experimental studies were performed: (i) in vitro analysis of the GLP-1R and downstream signaling pathway in human and rat choroid plexus, where GLP-1R expression was determined through mRNA analysis, immunostaining of human choroid plexus tissue sections, and fluorescently tagged exendin-4 binding to rat choroid plexus explants. The downstream signaling pathway was assessed in rat CPe cell culture by measuring cAMP generation and Na+ K+ ATPase activity. In vivo studies to determine the efficacy of exendin-4 to reduce ICP were conducted in (ii) healthy rats and (iii) a pathological model of raised ICP, a rat model of hydrocephalus. The sample size (n = 4 to 9 per experimental group) for the in vivo studies was based on the resource equation because the effects size was unknown. Exact numbers for each experiment are included below and in the figure legends. The investigators were not blinded when conducting or evaluating the experiments, and the rats were randomly assigned to the treatment and control groups.

Human tissue

Human choroid plexus samples were obtained from the Parkinson’s UK Brain Bank at Imperial College, London under the ethical approval of the Wales Research Ethics Committee (reference no. 08/MRE09/31+5). Informed consent was obtained for the use of postmortem tissue for research. Samples were stored in RNAlater at −80°C before being processed for quantitative polymerase chain reaction (qPCR) following the protocol stated in Supplementary Materials and Methods. Pooled human pancreas (540023), heart (540011), and ovary (540071) RNA was purchased from Agilent Technologies. Fresh choroid plexus samples were fixed in 4% formaldehyde before embedding in paraffin wax.

Experimental animals

For the in vitro work, female Sprague-Dawley rats (150 to 200 g; Charles River Laboratories) were used at the University of Birmingham in accordance with the Animals and Scientific Procedures Act 1986, licensed by the UK Home Office and approved by the University of Birmingham Ethics Committee. For the in vivo studies, which were conducted in Rigshospitalet-Glostrup, female Sprague-Dawley rats (150 to 250 g; Taconic) were housed in groups of four and kept under a 12-hour light/dark cycle with free access to food and water. All experimental procedures were approved by the Danish Animal Experiments Inspectorate (license no. 2014-15-0201-00256 and 2012-15-2934-00283). After treatments and surgical procedures, the rats were monitored daily for any adverse effects. Female rats were used to ensure that the results were relevant to conditions such as idiopathic intracranial hypertension.

Daily subcutaneous injections of exendin-4 in normal conscious rats. On day 0, the epidural ICP probe was implanted, and the animal was allowed to recover. On days 2, 4, and 6, for the ICP recordings, the rats were sedated with midazolam (2.5 mg/kg, subcutaneously) in an infusion cage (Instech Laboratories), which had a swirl lever arm to ensure unhindered movement. A stable baseline ICP reading was recorded for around 30 min before the rats received a subcutaneous injection of either saline (n = 9) or exendin-4 (20 μg/kg) (n = 9). ICP was recorded for a further 60 min after which the rat was returned to its normal cage. The daily subcutaneous injections of saline or exendin-4 were performed at similar times of the day for each rat.

Intracerebroventricular injection of exendin-4 in anesthetized rats. To determine whether the effects of exendin-4 on ICP were due to central activity, we fitted the rats with an intracerebroventricular cannula at the same time as the epidural ICP probe implantation and allowed the rat to recover. Subsequent ICP recordings during exendin-4 treatment were done under anesthesia. A stable baseline ICP reading was recorded for around 30 min before the following treatments were then administered intracerebroventricularly in a counterbalance design: (i) 1 μl of saline (n = 8) and (ii) exendin-4 (0.3 μg/μl) (n = 6). ICP was recorded for a further 60 min after which the rat was allowed to recover. Injection treatments were separated by 2 to 3 days.

Continuous intracerebroventricular infusion of exendin 9-39 with subcutaneous injection of exendin-4 in conscious rats. To determine whether the effects of exendin-4 on ICP are through the GLP-1R, we fitted the rats with an osmotic pump attached to an intracerebroventricular cannula containing either saline or exendin 9-39 at the same time as the epidural ICP probe implantation. On day 2, the rats were sedated, and a stable baseline was recorded before a subcutaneous injection of either saline or exendin-4 (20 μg/kg). ICP was then recorded for a further 60 min. The rats were therefore assigned to three treatment groups: (i) saline-filled osmotic pump with subcutaneous injection of exendin-4 (ICV saline + SC exendin-4; n = 6), (ii) exendin 9-39–filled osmotic pump with subcutaneous injection of saline (ICV exendin 9-39 + SC saline; n = 5), and (iii) exendin 9-39–filled osmotic pump with subcutaneous injection of exendin-4 (ICV exendin 9-39 + SC exendin-4; n = 6).

Exendin-4 dose response and time course experiment. Rats underwent the same procedure as outlined in the first experiment. For the dosing experiment, the rats were given exendin-4 (1 μg/kg, n = 6; 3 μg/kg, n = 6; or 5 μg/kg, n = 6), and ICP was recorded for 3 hours. For the time course experiment, rats were given either saline (n = 18 for ICP data but only 4 were used for choroid plexus analysis) and ICP was recorded for 24 hours or exendin-4 (5 μg/kg) and ICP was recorded for 6 (n = 6), 12 (n = 6), and 24 hours (n = 12 for ICP data but only 6 were used for choroid plexus analysis). After each time point, the rats were sacrificed with an overdose of euthatal and transcardially perfused with ice-cold phosphate-buffered saline. The choroid plexus was then dissected, frozen immediately, and stored at −80°C for qPCR and Western blot analysis (described in detail in Supplementary Materials and Methods).

Subcutaneous injection of exendin-4 in conscious hydrocephalic rats. We used the kaolin model of hydrocephalus as our model of raised ICP. On day 0, the rats received an injection of kaolin to induce hydrocephalus and were allowed to recover. On days 6 to 8, the rats were fitted with an epidural ICP probe and were then allowed to recover in the infusion cages still connected to the transducer so that the ICP could be continuously measured overnight to establish raised ICP. The following morning, after establishing that the baseline ICP reading was stable, the rats received a subcutaneous injection of either saline (n = 6; n = 4, >10 mmHg) or exendin-4 (20 μg/kg) (n = 6; n = 4, >10 mmHg). ICP was then recorded for a further 60 min.

Statistical analysis

Values are represented as means and SEM. Most of the data were analyzed using GraphPad Prism software; however, the time course experiment with exendin-4 (5 μg/kg) was analyzed using SPSS because of missing data points. For the enzyme-linked immunosorbent assay cAMP analysis, the nonparametric Kruskal-Wallis test was used followed by Mann-Whitney test (two-tailed) with the appropriate adjustment for multiple comparisons (Bonferroni). t test or one-way ANOVA (followed by Tukey post hoc test) was used for the comparison of qPCR, Western blot, and Na+ K+ ATPase activity. Two-way ANOVA with Sidak’s multiple comparisons test was used for the comparison of ICP between two groups over a period of time. Values were considered statistically significant when P values were *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Individual level data are included in table S1.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/9/404/eaan0972/DC1

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.

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

REFERENCES AND NOTES

  1. Funding: A.J.S. was funded by a National Institute for Health Research Clinician Scientist Fellowship (NIHR-CS-011-028) and by the Medical Research Council (MRC), UK (MR/K015184/1). D.J.H. was supported by Diabetes UK R.D. Lawrence (12/0004431), European Foundation for the Study of Diabetes/Novo Nordisk Rising Star and Birmingham Fellowships, a Wellcome Trust Institutional Support Award, an MRC Project Grant (MR/N00275X/1), and a European Research Council Starting Grant (OptoBETA; 715884). This work was supported by an MRC confidence-in-concept grant, the West Midlands Neuroscience Teaching and Research Fund, and the University of Birmingham Research Development Fund. Author contributions: A.J.S. was responsible for the study concept. H.F.B., A.M.G., D.J.H., and A.J.S. conceived and designed the experiments; H.F.B. conducted the in vitro experiments (immunohistochemistry, Na+ K+ ATPase activity assay, cAMP assay, rat qPCR and Western blot, and FLEX analysis); C.S.J.W. performed human qPCR and cAMP assays; A.M.G., M.S.U., and J.L.M. contributed to the immunohistochemistry data; H.F.B., M.S.U., J.L.M., and S.M.H. performed the ICP recordings; H.F.B. and M.S.U. analyzed the data; and H.F.B., M.S.U., A.M.G., D.J.H., R.H.J., and A.J.S. co-wrote the manuscript. All authors reviewed the final version. Competing interests: A.J.S. is an inventor and the University of Birmingham an applicant on patent application no. PCT/GB2015/052453 related to this work entitled “Elevated intracranial pressure treatment.” R.H.J. has given lectures for Pfizer, Berlin-Chemie, Norspan, Merck, and Autonomic Technologies and has been a member of the advisory boards of Autonomic Technologies, Medotech, and ElectroCore. All other authors declare that they have no competing interests.
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