Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy

Abstract

Hypoxic-ischaemic encephalopathy remains a global health burden. Despite medical advances and treatment with therapeutic hypothermia, over 50% of cooled infants are not protected and still develop lifelong neurodisabilities, including cerebral palsy. Furthermore, hypothermia is not used in preterm cases or low resource settings. Alternatives or adjunct therapies are urgently needed. Exendin-4 is a drug used to treat type 2 diabetes mellitus that has also demonstrated neuroprotective properties, and is currently being tested in clinical trials for Alzheimer's and Parkinson's diseases. Therefore, we hypothesized a neuroprotective effect for exendin-4 in neonatal neurodisorders, particularly in the treatment of neonatal hypoxic-ischaemic encephalopathy. Initially, we confirmed that the glucagon like peptide 1 receptor (GLP1R) was expressed in the human neonatal brain and in murine neurons at postnatal Day 7 (human equivalent late preterm) and postnatal Day 10 (term). Using a well characterized mouse model of neonatal hypoxic-ischaemic brain injury, we investigated the potential neuroprotective effect of exendin-4 in both postnatal Day 7 and 10 mice. An optimal exendin-4 treatment dosing regimen was identified, where four high doses (0.5 µg/g) starting at 0 h, then at 12 h, 24 h and 36 h after postnatal Day 7 hypoxic-ischaemic insult resulted in significant brain neuroprotection. Furthermore, neuroprotection was sustained even when treatment using exendin-4 was delayed by 2 h post hypoxic-ischaemic brain injury. This protective effect was observed in various histopathological markers: tissue infarction, cell death, astrogliosis, microglial and endothelial activation. Blood glucose levels were not altered by high dose exendin-4 administration when compared to controls. Exendin-4 administration did not result in adverse organ histopathology (haematoxylin and eosin) or inflammation (CD68). Despite initial reduced weight gain, animals restored weight gain following end of treatment. Overall high dose exendin-4 administration was well tolerated. To mimic the clinical scenario, postnatal Day 10 mice underwent exendin-4 and therapeutic hypothermia treatment, either alone or in combination, and brain tissue loss was assessed after 1 week. Exendin-4 treatment resulted in significant neuroprotection alone, and enhanced the cerebroprotective effect of therapeutic hypothermia. In summary, the safety and tolerance of high dose exendin-4 administrations, combined with its neuroprotective effect alone or in conjunction with clinically relevant hypothermia make the repurposing of exendin-4 for the treatment of neonatal hypoxic-ischaemic encephalopathy particularly promising.

Figure 1

Schematic of different exendin-4 treatment regimens. (A) Schematic of exendin-4 dosing regimen in the late preterm hypoxia-ischaemia model (P7). (B) Schematic of combining clinically relevant hypothermia and exendin-4 treatment in the term hypoxia-ischaemia model (P10). Mouse by Iconic from the Noun Project.

Figure 2

GLP1R expression in the human preterm and murine brain across different developmental ages. (A) Immunofluorecence and scanning confocal microscopy studies demonstrating GLP1R is expressed in neurons (NeuN) in the mouse brain at 10 weeks (adult), P10 and P7, (B) with only small colocalization with astrocytes (GFAP) at 10 weeks of age, and (C) no co-localization with microglia (CD68) cells in any of the different developmental ages. The first row of micrographs (A–C) show negative staining for GLP1R antibody. (D) Immunofluorescence studies of post-mortem human preterm brain tissue shows co-localization of GLP1R expression in neurons (NeuN) in the frontal lobe and hippocampus Ammon’s horn, with the first row of micrographs for each human brain region containing negative staining for GLP1R antibody. A median filter was applied to the images to reduce noise. (E) Relative quantification of GLP1R expression by quantitative PCR in different brain regions at 10 weeks, (F) P10 and (G) P7 (n = 6 per age group). CBL = cerebellum; CTX = cortex; HIP = hippocampus; HPT = hypothalamus; MDL = medulla; OB = olfactory bulbs; THL = thalamus. Scale bar = 20 µm in C; 10 µm in D.

Figure 3

Evaluation of optimal exendin-4 dose and time treatment regimen in the P7 late preterm model. (A) Representative whole brain micrographs of the different treatment groups: saline (n = 14, eight male and six females); one high dose exendin-4 (n = 14, eight male and six female), four high doses exendin-4 (n = 14, seven male and seven female) and four low-doses exendin-4 (n = 14, seven male and seven female) started immediately after hypoxia-ischaemia, and four high doses exendin-4 initiated 2 h after hypoxia-ischaemia (n = 14, seven male and seven female). (B) Effect on ipsilateral hemispheric tissue loss of different dose regimen started immediately after hypoxia-ischaemia and (C) delayed start of exendin-4 treatment. (D) Weight gain over a 48 h period following immediate exendin-4 administration at the different doses and (E) different time. Data presented as individual animals ± SEM and analysed using Kruskal-Wallis Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001. Scale bar = 2 mm. EX-4 = exendin-4; HI = hypoxia-ischaemia.

Figure 4

Exendin-4 treatment reduces cell death 48 h after P7 hypoxia-ischaemia. (A) Hippocampus micrograph representation with high magnification inserts (×40), of saline, immediate and 2 h delayed four high dose exendin-4 treatment. (B) Overall and individual (C) isocortex, (D) pyriform cortex, (E) external capsule, (F) hippocampus, (G) striatum and (H) thalamus brain regions quantification of TUNEL+ cell death 48 h after hypoxic-ischaemic injury across the three different treatment groups. Data presented as number of TUNEL+ cells per individual animal ± SEM and analysed using Kruskal-Wallis Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Scale bar = 400 µm. HI = hypoxia-ischaemia.

Figure 5

Exendin-4 suppresses glial cell activation 48 h after hypoxia-ischaemia. Overall and regional assessment of microglia activation (alphaM, αmβ2) through semi-quantitative score (A), and quantitative immunoreactivity analysis of astrocytes (GFAP) (B) and endothelial cells (ICAM1) (C). Data presented as individual animals ± SEM and analysed using Kruskal-Wallis Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 6

Exendin-4 administration blood analysis. (A) Blood glucose measurement at different time points in naïve and treated mice following high dose (0.5 µg/g) exendin-4 injection (n = 4 per group). (B) Weight measurement in naïve, saline and exendin-4 alone during the four high dose administration regimen. (C) Total white blood cell (WBC), (D) neutrophils, (E) lymphocytes, (F) monocytes, (G) eosinophils and (H) basophils counts, (I) haematocrit (HCT), (J) platelets, (K) red blood cells (RBC), (L) haemoglobin and (M) mean corpuscular volume (MCV) at the end of the treatment regimen. Data presented as individual animals or as mean ± SEM and analysed using Kruskal-Wallis Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. EX4 = exendin-4; SAL = saline.

Figure 7

Histopathological organ assessment. (A) Representative micrographs of haematoxylin and eosin stained brain, including hippocampus (hip) level and visceral organs: heart, liver, pancreas, spleen, lung and kidney showing no abnormal histopathology in naïve, saline (SAL) or exendin-4 (EX4) alone treatments. (B) Quantitative immunoreactivity threshold measurements of macrophages (CD68) in the brain, (C) heart, (D) spleen, (E) liver, (F) lung, (G) pancreas and (H) kidney for the different groups (n = 6 per group). Data presented as individual animals ± SEM and analysed using Kruskal-Wallis Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure 8

Synergistic enhanced neuroprotection following combined exendin-4 and hypothermia treatment in the term hypoxia-ischaemia model. (A) Whole brain representative micrographs of normothermia + saline control (NT SAL, n = 24, 10 male and 14 female), single treatments normothermia + exendin-4 (NT EX4, n = 25, 12 male and 13 female) and hypothermia + saline (HT SAL, n = 25, 14 male and 11 female), as well as combined hypothermia and exendin-4 treatment (HT EX4, n = 25, 12 male and 13 female) 7 days after P10 hypoxic-ischaemic injury. (B) Macroscopic score evaluation. (C) Weight gain across the different groups. (D) Overall infarct volume and (E) through different injury levels, where level 1 indicates the most anterior level, as assessed using MAP-2-stained sections from the different treatments. Data presented as individual animals or as mean ± SEM and analysed using Kruskal-Wallis Dunn’s test. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Scale bar = 2 mm.