Repetitive acute intermittent hypoxia increases growth/neurotrophic factor expression in non-respiratory motor neurons

Abstract

Repetitive acute intermittent hypoxia (rAIH) increases growth/trophic factor expression in respiratory motor neurons, thereby eliciting spinal respiratory motor plasticity and/or neuroprotection. Here we demonstrate that rAIH effects are not unique to respiratory motor neurons, but are also expressed in non-respiratory, spinal alpha motor neurons and upper motor neurons of the motor cortex. In specific, we used immunohistochemistry and immunofluorescence to assess growth/trophic factor protein expression in spinal sections from rats exposed to AIH three times per week for 10weeks (3×wAIH). 3×wAIH increased brain-derived neurotrophic factor (BDNF), its high-affinity receptor, tropomyosin receptor kinase B (TrkB), and phosphorylated TrkB (pTrkB) immunoreactivity in putative alpha motor neurons of spinal cervical 7 (C7) and lumbar 3 (L3) segments, as well as in upper motor neurons of the primary motor cortex (M1). 3×wAIH also increased immunoreactivity of vascular endothelial growth factor A (VEGFA), the high-affinity VEGFA receptor (VEGFR-2) and an important VEGF gene regulator, hypoxia-inducible factor-1α (HIF-1α). Thus, rAIH effects on growth/trophic factors are characteristic of non-respiratory as well as respiratory motor neurons. rAIH may be a useful tool in the treatment of disorders causing paralysis, such as spinal injury and motor neuron disease, as a pretreatment to enhance motor neuron survival during disease, or as preconditioning for cell-transplant therapies.

Keywords: BDNF; HIF-1; TrkB; VEGF; intermittent hypoxia; motor neuron.

Published by Elsevier Ltd.

Figures

Fig. 1

Representative images of BDNF immunostaining in primary motor cortex (M1), cervical (C7) and lumbar (L3) ventral horn spinal cord. 3 × wAIH increased BDNF protein expression in upper motor neurons in M1, especially in layer V (Boxes) (B) versus normoxic control (A). Higher magnification images from layer V are in the bottom of the images. Immunostaining also confirmed BDNF expression in presumptive alpha motor neurons (big arrow) and interneurons (small arrow heads) in cervical (C7) and lumbar (L3) segments, and its immunoreactivity is enhanced following 3 × wAIH (C–F). BDNF and NeuN (Neuronal marker)-positive neurons (white arrow head) in L3 in lamina nine ventral horn, where BDNF immunoreactivity was increased by 3 × wAIH (G–J), as confirmed by densitometry (K). Scale bars: A, B 400 µm, higher-magnification on figures A, B is 100 µm, C–F is 200 µm, G–J is 100 µm. Data are mean±1 SEM. *p < 0.05 versus normoxia.

Fig. 2

Photomicrographs of TrkB (A–D) and phospho-TrkB (E, F) in primary motor cortex (layer V of M1) and C7 ventral horn. TrkB immunoreactivity (dark brown) was counterstained with cresyl-violet (blue) to determine the localization of Betz cells in layer V of the primary motor cortex (A, B) and in alpha motoneurons of C7 ventral horn (C, D). TrkB receptor protein expression and phospho-TrkB were upregulated in motor neurons of M1 and C7 following 3 × wAIH treatment (B, D, F, H) compared to normoxic controls (A, C, E, G). Higher-magnification images (100 µm; insets) from C7 (C, D, G, H) and M1 (E, F) are shown in enlarged boxes. Scale bars: A–F 200 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Representative images of VEGF immunostaining in layer V of M1 and cervical (C7) and lumbar (L3) ventral horn. VEGF immunostaining is increased in M1, C7 and L3 ventral horn after 3 × wAIH treatment (D–F) versus normoxic control (A–C). VEGF and NeuN-positive neurons (white arrow head), where VEGF immunoreactivity was increased following 3 × wAIH (G–J), as confirmed by densitometry (K). Scale bar for A–F: 200 µm; G–J is 100 µm. Data are mean±1 SEM. **p < 0.001 versus normoxia.

Fig. 4

Photomicrographs of VEGF receptor-2 (VEGFR-2/KDR) immunostaining in layer V of M1 and in C7 and L3 ventral horn. 3 × wAIH increased VEGF receptor protein expression in layer V of primary motor cortex (M1) and in alpha-motor neurons of C7 and L3 ventral horn (D–F) versus normoxic control (A–C). Scale bars for A–F: 200 µm.

Fig. 5

Photomicrographs of HIF-1α immunostaining in layer V of cortical M1 and in C7 and L3 ventral horns. Repetitive AIH increased HIF-1α protein expression in motor neurons in layer V of the primary motor cortex (M1) and in alpha motor neurons of C7 and L3 ventral horns (D–F) versus normoxic control (A–C). HIF-1α and NeuN-positive neurons (white arrow head), where HIF-1α immunoreactivity was increased following 3 × wAIH (G–J), as confirmed by densitometry (K). Scale bars for A–F: 200 µm; G–J is 100 µm. Data are mean±1 SEM. *p < 0.05 versus normoxia.