Brain Metabolism Alterations Induced by Pregnancy Swimming Decreases Neurological Impairments Following Neonatal Hypoxia-Ischemia in Very Immature Rats

Abstract

Introduction: Prematurity, through brain injury and altered development is a major cause of neurological impairments and can result in motor, cognitive and behavioral deficits later in life. Presently, there are no well-established effective therapies for preterm brain injury and the search for new strategies is needed. Intra-uterine environment plays a decisive role in brain maturation and interventions using the gestational window have been shown to influence long-term health in the offspring. In this study, we investigated whether pregnancy swimming can prevent the neurochemical metabolic alterations and damage that result from postnatal hypoxic-ischemic brain injury (HI) in very immature rats. Methods: Female pregnant Wistar rats were divided into swimming (SW) or sedentary (SE) groups. Following a period of adaptation before mating, swimming was performed during the entire gestation. At postnatal day (PND3), rat pups from SW and SE dams had right common carotid artery occluded, followed by systemic hypoxia. At PND4 (24 h after HI), the early neurochemical profile was measured by ¹H-magnetic resonance spectroscopy. Astrogliosis, apoptosis and neurotrophins protein expression were assessed in the cortex and hippocampus. From PND45, behavioral testing was performed. Diffusion tensor imaging and neurite orientation dispersion and density imaging were used to evaluate brain microstructure and the levels of proteins were quantified. Results: Pregnancy swimming was able to prevent early metabolic changes induced by HI preserving the energetic balance, decreasing apoptotic cell death and astrogliosis as well as maintaining the levels of neurotrophins. At adult age, swimming preserved brain microstructure and improved the performance in the behavioral tests. Conclusion: Our study points out that swimming during gestation in rats could prevent prematurity related brain damage in progeny with high translational potential and possibly interesting cost-benefits. HIGHLIGHTS - Prematurity is a major cause of neurodevelopmental impairments;- Swimming during pregnancy reduces brain damage after HI injury;- Pregnancy is an important but underestimated preventive window.

Keywords: brain; hypoxia-ischemia; magnetic resonance imaging; neuroprotection; pregnancy swimming; prematurity.

Figure 1

Experimental timeline. GD, Gestational Day; HI, Hypoxia-Ischemia; PND, Postnatal day; DTI, Diffusion Tensor Imaging; NODDI, Neurite Orientation Dispersion and Density Index; SE, Sedentary; SW, Swimming.

Figure 2

Effect of pregnancy swimming on the pup's protein levels in right cortex extracts 24 h following HI (PND4). Bars graph represent immunoblots of (A) neurons (NeuN) and migrating neurons Doublecortin (DCX), oligodendrocytes progenitor (NG2) and microglia (Iba-1); (B) cell death (ccaspase 3 and fractin) and inflammation (CD11b), (C) neurotrophins VEGF and BDNF and the TRK-B receptor and (D) astrogliosis (GFAP), glutamate receptor GLT-1 and glutamine synthetase enzyme in the four experimental groups: sedentary sham (SESH), sedentary hypoxic-ischemic (SEHI), swimming sham (SWSH) and swimming hypoxic-ischemic (SWHI). WB results are plotted normalized to SESH level expression (100%) (mean ± SEM). Significance testing was determined using one-way ANOVA followed by Duncan's post hoc and was performed on Actin or βIII-tubulin normalized data. *HI vs. its respective SH group, Significance accepted when p < 0.05.

Figure 3

Effect of pregnancy swimming on the pup's protein levels on the right hippocampus extracts 24 h following HI (PND4). Bars graph represent immunoblots of (A) neurons (NeuN) and migrating neurons Doublecortin (DCX), oligodendrocytes progenitors (NG2) and microglia (Iba-1); (B) cell death (ccaspase 3 and fractin) and inflammation (CD11b), (C) neurotrophins VEGF and BDNF and the TRK-B receptor and (D) astrogliosis (GFAP), glutamate receptor GLT-1 and glutamine synthetase enzyme in the four experimental groups: sedentary sham (SESH), sedentary hypoxic-ischemic (SEHI), swimming sham (SWSH) and swimming hypoxic-ischemic (SWHI). WB results are plotted normalized to SESH level expression (100%) (mean ± SEM). Significance testing was determined using one-way ANOVA followed by Duncan's post-hoc and was performed on Actin or βIII-tubulin normalized data. *HI vs. its respective SH group. ^#SWHI vs. SESH and SEHI. Significance accepted when p < 0.05.

Figure 4

DT-MRI microstructural white matter alterations at PND4. Histograms of mean values ± SEM of the DTI derived parameters at PND4 in the cortex (A), and external capsule (B). Axial Diffusivity (AD), Radial Diffusivity (RD), Median diffusivity (MD), fractional anisotropy (FA) and NODDI estimates: intraneurite volume fraction (f_icvf), cerebrospinal volume fraction (f_iso) and orientation dispersion index (ODI) for SESH, SEHI, SWSH and SWHI groups. *SWHI vs. SWSH. ^#SESH vs. all other groups.

Figure 5

Water Maze performance during the 5 days of training (left upper panel). (Right upper panels) - performance on the probe trial. Data are expressed as mean ± SEM (n = 8–16). Lower panels show the representative plots of the Probe Trial. The results were analyzed by two-way ANOVA followed by Duncan's post-hoc test. Significance was accepted when p < 0.05. *SEHI vs. SESH.

Figure 6

Using ex vivo DT-MRI long-term microstructural white matter alterations were analyzed at PND60 following neonatal HI. White matter microstructure is partially restored by pregnancy swimming. Histograms of mean values ± SEM of DTI derived parameters: Axial Diffusivity (AD), Radial Diffusivity (RD), Median diffusivity (MD), fractional anisotropy (FA) and NODDI estimates: intraneurite volume fraction (ficvf), cerebrospinal volume fraction (fiso) and orientation dispersion index (ODI) in the external capsule (A), cerebral cortex (B), and corpus callosum (C) for SESH, SEHI, SWSH and SWHI rats at P60. *SEHI vs. SESH, ^#SESH vs. SWSH, ^§SEHI vs. SWHI; p < 0.05.

Figure 7

Protein expression of MBP, NeuN, GFAP, BDNF, TRK-β, and VEGF in right cortex (left upper panel) and hippocampus (right upper panel) at PND60. Lower panels: representative immunoblots of cortex (left lower) and hippocampus (right lower). WB results are plotted normalized to the SESH group level expression (100%) (mean ± SEM). Significance testing was determined using one-way ANOVA followed by Duncan's post-hoc using Actin or βIII-tubulin as normalizer. *SESH vs. SEHI. Significance accepted when p < 0.05.