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. 2016 Dec 29;37(7):1269–1278. doi: 10.1007/s10571-016-0459-8

RETRACTED ARTICLE: Metformin Attenuates Cognitive Impairments in Hypoxia–Ischemia Neonatal Rats via Improving Remyelination

Boxiang Qi 1, Libao Hu 1, Lei Zhu 1, Lei Shang 1, Liping Sheng 1, Xuecheng Wang 1, Na Liu 1, Nana Wen 1, Xiaohe Yu 2, Qihong Wang 2, Yujia Yang 2,✉
PMCID: PMC11482152  PMID: 28035478

Abstract

Perinatal hypoxia–ischemia (H/I) causes brain injury and myelination damage. Finding efficient methods to restore myelination is critical for the recovery of brain impairments. By applying an H/I rat model, we demonstrate that metformin (Met) treatment significantly ameliorates the loss of locomotor activity and cognition of H/I rat in the Morris water maze and open field task tests. After administration of Met to H/I rat, the proliferation of Olig2+ oligodendrocyte progenitor cells and the expression of myelin basic protein are obviously increased in the corpus callosum. Additionally, the myelin sheaths are more compact and the impairments are evidently attenuated. These data indicate that Met is beneficial for the amelioration of H/I-induced myelination and behavior deficits.

Keywords: Perinatal hypoxia–ischemia, Metformin, Cognitive deficit, Oligodendrocyte progenitor cells

Introduction

Perinatal hypoxia–ischemia (H/I) can lead to neurodevelopmental injury and disability in the preterm infants (Tichauer et al. 2009). A major pathological feature in the H/I brain is the impairments of white matter (WM) (Carty et al. 2011). WM occupies approximately a half of the brain volume. The blood supply in the WM is small and has no or little collateral circulation, which makes WM susceptible to hypoxia–ischemia injury (Arai and Lo 2009; Shereen et al. 2011). H/I-induced WM damage may result in demyelination (Drobyshevsky et al. 2007; Mao et al. 2012). Demyelinating lesions can lead to axonal degeneration. Demyelination and axonal loss in different cortical regions might cause distinct neuropathies (Van Asseldonk et al. 2003). Although people attach importance to the demyelination diseases, there is no efficient therapy for H/I-induced brain injury. Investigations on effective interventions and remyelination mechanisms are critical to the treatments of neurological deficits.

In the central nervous system, mature oligodendrocytes (OLs) extend processes to touch and wrap neuronal axons, forming myelin sheath (Fulton et al. 2011; Kuo et al. 2010; Morello et al. 2011). All the steps are well regulated (Morello et al. 2011). Intact myelin sheaths play important roles in neuronal electrical propagation. Loss of myelin sheath may cause different diseases, such as palsy and sclerosis (Fancy et al. 2011). OLs mainly derive from oligodendrocyte progenitor cells (OPCs) during embryonic periods, sensitive time for leukomalacia (Manning et al. 2008; Talos et al. 2006). OPCs are highly susceptible to hypoxia. The generation and maturation of OLs are highly relevant to the integrity of brain organization and critical for myelin reformation in demyelination disorders. The degeneration of OPCs and OLs lead to myelination failure and WM impairments (Riddle et al. 2012).

Metformin (Met) is a biguanide drug widely used to treat diabetes mellitus (Rojas and Gomes 2013). It has been demonstrated that Met benefits for oxidative stress modulation and angiogenesis (Abd-Elsameea et al. 2014; Venna et al. 2014). It has also been suggested to apply Met for ischemic stroke therapy (Wang et al. 2012). Met can inhibit cell apoptosis in kinds of tissues such as liver and brain (Ashabi et al. 2015; Vytla and Ochs 2013; Viollet and Foretz 2013; Kristensen et al. 2013). Met exhibits neuroprotective effects in Alzheimer’s disease and other neurodegenerative diseases (Ma et al. 2007; Sridhar et al. 2015). However, whether Met promotes myelin repair in demyelinating diseases remains to be unraveled.

Through perinatal hypoxia–ischemia (H/I) rat models, this study shows that Met administration significantly ameliorates the defects of locomotor and cognitive activities. Meanwhile, the proliferation of OPCs and the level of myelin basic protein (MBP) are evidently increased in the corpus callosum (CC). The axon-wrapped structure is obviously recovered. These data suggest that Met treatment may be clinically useful for the therapy of H/I-induced demyelination impairments.

Materials and Methods

The Establishment of Perinatal Hypoxia–Ischemia (H/I) Rat Model

H/I was produced as described previously (Buller et al. 2008; Qu et al. 2014). On P3, pups were anesthetized with isoflurane via the pup’s snout. The right common carotid artery was carefully isolated from surrounding tissue and ligated twice. The wound was sutured and pups were allowed to recover for 2 h before being exposed to 6% O2 (94% N2 saturation) at 37 °C for 90 min in a humidified chamber. The mortality rate was <30%, and we chose 12 rat pups from each group for the following trials. The control group underwent the same procedures but the carotid artery was not ligated and they were exposed to room-air instead of hypoxia. Rat pups on postnatal day 3 (P3) obtained from the center of experiment animal of Xuzhou Medical College were randomly divided into four groups: (1) control-operated group; (2) P3 H/I; (3) P3 H/I, Met (50 mg/kg) group; and (4) P3 H/I, vehicle group (dH2O/0.1% Tween-80). All experiments were performed in accordance with the guidelines regarding the care and use of animals for experimental procedures and had institutional approval from both the Provisions and General Recommendations of Chinese Experimental Animal Administration Legislation and Xuzhou Medical College. The rats in the home cage were under a 12:12 h light:dark cycle, with food and water freely available throughout the study.

Met Administration

Met in distilled water was administered intragastrically once a day at the doses of 50 mg/(kg per day) from 2 h after H/I to the day the rats were sacrificed. The vehicle control group was given in distilled water (dH2O/0.1% Tween-80) without Met.

Morris Water Maze (MWM) Test

MWM test is used to evaluate the ability of spatial learning and memory (Tsai et al. 2011). Six weeks after P3 H/I, pups were tested on the MWM at four trials per day for five days to test spatial reference memory. The MWM, with a hidden platform remaining in a fixed location, was filled with water of 23 ± 2 °C. During the test, the rat was placed in the maze from the North, South, East, or West location, and it was given 60 s to locate the hidden platform. The trial was not terminated until the rat found the platform. After staying on the platform for 15 s, the rat was placed into its heated cage for the next trial. To evaluate whether rats localized the platform in a 60-s probe trial, on day 5, the platform was removed. For each trial, a camera suspended above the maze was used to track each rat’s path and a tracking system (Ethovision 3.1, Noldus Instruments) was used for analyzing each rat’s tracing. Swim path distance (cm) and escape latency (s) to the platform were used to assess MWM performance. For probe trial data, the number of crossing previous platform location in the target quadrant was recorded.

Open Field Test

The open field apparatus consists of a square, opaque acrylic container and a video camera fixed 1 m above the arena tracking the rats’ movement. A computerized tracking system analyzed the images and measured speed and distance of movement. Tests were performed in the breeding room after water maze test. Rats were placed in the dimly lit room for 1 h before testing to acclimatize to the new environment. The individual rat was placed in the middle of the chamber for each trial. After 1-min adaptation, the behavior of each rat was recorded for 5 min. During the interval between trials, rat was returned to its home cage in the same room and the open field was wiped clean with a slightly damp cloth. The number of rearing events, grooming sessions, total distance, and speed traveled was recorded. To assess the anxiety of rats in the open field, the time spent in the central area was also recorded.

Western Blot

After reperfusion under anesthesia, rats were decapitated immediately and then the CC was removed and homogenized in 1:10 (w/v) ice-cold homogenization buffer. After centrifuging at 12,000g for 15 min at 4 °C, the supernatant was collected for protein concentration test. Equal amounts of proteins (100 mg/lane) were separated by 10% SDS-PAGE and then electrotransfered onto a nitrocellulose membrane by a semidry blotting system (Amersham, Buckinghamshire, United Kingdom). After being blocked for 3 h in PBS consisting of 0.1% Tween-20 and 3% bovine serum albumin (BSA), membranes were incubated overnight at 4 °C with primary antibodies in PBS with 0.1% Tween 20 containing 1% BSA. Anti-MBP antibody was purchased from Abcam, while anti-Olig2 antibody and anti-Id2 antibody were purchased from Santa Cruz Biotechnology. Being washed for three times, membranes were incubated with alkaline phosphatase-conjugated secondary antibodies in PBS with 0.1% Tween 20 for 2 h. Immunoreactivity was detected by NBT/BCIP assay kit (Promega, Madison, WI). The densities of the bands on the membrane were scanned and analyzed with an image analyzer (LabWorks Software, Upland, CA).

Electron Microscopy

The preparation of material followed standard electron microscopy protocols. After elimination of the blood by normal saline, the rats were anesthetized with pentobarbital and perfused with buffered 2.5% glutaraldehyde through the left ventricle. A small brain white matter block (1 × 1 × 1 mm3) from a similar area of the left hemisphere above the left paracele, an area <1 mm2 posterior from Bragma site and >1 mm2 from midline, was removed and post-fixed in 3% glutaraldehyde (0–4 °C). Then samples were incubated with 1% osmic acid for 3 h after rinsing, and dehydrated with acetone and embedded in epoxy resin. Ultrathin sections (60 nm) were made from the resin-embedded samples and stained with uranyl acetate and lead citrate prior to examination by transmission electron microscopy (TEM).

BrdU Labeling and Immunohistochemistry Staining

To evaluate the newly generated cells in the brain, rats of each group were intraperitoneally injected with BrdU (100 mg/kg, sigma) 12 h after H/I injury. Immunohistochemistry staining for brain tissue was performed on coronal ice-cold sections (40 μm){Zhang, 2016 #470}. Briefly, sections were blocked with 0.01 M phosphate-buffered saline (PBS) containing 5% goat serum for 1 h at RT. Then the sections were incubated with anti-MBP antibody or anti-Olig2 antibody (Abcam, Cambridge, UK). For BrdU immunostaining, sections were pretreated in 2 M HCl for 60 min at 37 °C followed by extensive rinses in 0.1 M borate buffer, pH 8.5, and with PBS/0.1% Triton X-100. Afterwards, tissue sections were blocked and incubated with anti-BrdU antibody (Abcam, Cambridge, UK). After treatment with primary antibodies, sections were rinsed with PBS and incubated for 2 h at room temperature with the appropriate fluorescent secondary antibody. Samples were observed under fluorescence microscopy (Olympus, Japan).

Statistical Evaluation

Western blot and histology examination were used to examine the kinds of samples that were obtained from four independent rats in each group. Data were presented as mean ± SD. Statistical significance between groups was carried out by one-way ANOVA followed by the least significant difference test or Newman–Keuls post hoc test. In all cases, p < 0.05 was considered significant.

Results

Metformin Attenuates Behavior Deficits in the H/I Rats

To evaluate roles of metformin (Met) on locomotor and exploratory activities, we used open field test (Walsh and Cummins 1976). Among the test, a total of five parameters were analyzed: total moving distance, score of rearing/leaning, average speed, time in corner, and grooming number. Locomotion was represented by the total distance and average speed. Exploratory activity was indicated by the score of rearing/leaning, and anxiety level was reflected by the time in corner and grooming number (Pietrelli et al. 2012). Compared with the control rats, the H/I rats had reduced traveling distance, slower moving speed, lower score of rearing/leaning, less time in corner, and grooming number (Fig. 1a–e, p < 0.05). While the treatment with vehicle had no effects on these parameters, all the values were improved in the Met treatment group (Fig. 1a–e, p < 0.05).

Fig. 1.

Fig. 1

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Metformin ameliorates impairments of locomotor and exploratory activities. Rats were divided into four groups (control group, H/I group, H/I plus Met group, H/I plus vehicle group). We measured distance (a), speed (b), rearing (c), time in corner (d), and grooming (e). All the values were presented as mean ± SD a p < 0.05, relative to control group. b p < 0.05, relative to H/I group. N = 8 for each group

Morris water maze (MWM) is usually applied to investigate spatial learning and memory (Wen et al. 2014). During the MWM test, all the rats were trained for 4 days and the escape latency was recorded every day. Normally, the escape latency decreased with increasing training days. The latency of H/I rats was longer than that of control rats. Met conditioning significantly reduced the H/I rats’ latency, and the vehicle treatment had no impacts on the latency (Fig. 2a, b, p < 0.05). On day 5 of the MWM test, the platform was removed. The distance rats traveled, the time rats spent, and the number rats crossed were recorded and compared among the different groups. We found that control rats showed a preference in the zone where the previous platform located (Fig. 3a–d), suggesting a spatial reference memory. This preference disappeared in the H/I group (Fig. 3a–d, p < 0.05). The percentage of distance and time and the number of crossing were recovered in the H/I plus Met group (Fig. 3a–d, p < 0.05).

Fig. 2.

Fig. 2

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Metformin attenuates spatial learning deficits. Rats were divided into control group, H/I group, H/I plus Met group, and H/I plus vehicle group. a Representative images for tracing rats’ swimming track. b The latency (time to reach the hidden platform) in each group was presented as mean ± SD. Data were shown as mean ± SD. a p < 0.05, relative to control group. b p < 0.05, relative to H/I group. N = 8 for each group

Fig. 3.

Fig. 3

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Metformin benefits memory recovery. Rats were divided into control group, H/I group, H/I plus Met group, and H/I plus vehicle group. On the 5th day, the platform was removed for the memory test. a Representative images of rats’ swimming track recordings in the water maze in each group. b, c The percentage of distance and time in the zone where the platform was located were calculated as mean ± SD. d The number that rats crossed the previous platform location in each group was presented as mean ± SD. a p < 0.05, relative to control group. b p < 0.05, relative to H/I group. N = 8 for each group

Metformin Increases the Proliferation of OPCs in the H/I Rats

Perinatal hypoxia–ischemia (H/I) leads to numerous neurons’ demyelination, and the proliferation of OPCs improves the myelination of neurons (Qu et al. 2014). Olig2, a basic helix-loop-helix transcription factor, is an OPC marker and critical for OLs specification and differentiation (Zhu et al. 2012). BrdU, a thymidine analog, can incorporate into replicating DNA and be an indicator of cell proliferation. To reveal whether Met could stimulate OPCs’ proliferation, we examined the expression of BrdU and Olig2 in the CC. Compared with the control group, Olig2+/BrdU+ cells were less in the H/I CC (Fig. 4a, b, e). While H/I rats with Met treatment contained more Olig2+/BrdU+ cells in the CC, the vehicle treatment made no change to the cell number (Fig. 4c, e). The western blot data were consistent with the immunostaining results. Olig2 protein decreased in the H/I group (Fig. 5a, b, p < 0.05). Met treatment significantly made the protein level increase, but vehicle administration had no effects (Fig. 5a, b, p < 0.05).

Fig. 4.

Fig. 4

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Metformin promotes the proliferation of OPCs in vivo. Olig2 (green) and BrdU (red) were double immunostained at P30. a–d Representative images in the control group (a), H/I group (b), H/I plus Met group (c), H/I plus vehicle group (d), respectively. (e) The quantification of Olig2 +/BrdU + cells in each group was compared and showed as mean ± SD. Bars, 30 μm (Color figure online)

Fig. 5.

Fig. 5

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Metformin treatment up-regulates Olig2 protein level. Rats were divided into control group, H/I group, H/I plus Met group, and H/I plus vehicle group. After reperfusion, total Olig2 protein was analyzed by western blot. a Immunoblot bands in each group. b The relative bands’ intensity in each group was compared. Data were given as mean ± SD. a p < 0.05, relative to control group. b p < 0.05, relative to H/I group. N = 8 for each group

Metformin Improves MBP Expression in the H/I Rats

To explore whether Met improves neurons’ remyelination, we detected the expression of myelin basic protein (MBP), a mature OL marker. Compared with the control brains, the H/I CC was rarefactional and the MBP staining was evidently lower (Fig. 6a, b). After Met treatment, the staining intensity of MBP was obviously stronger in the H/I CC (Fig. 6b, c). In contrast with this, the vehicle administration made the MBP staining no change in the injured CC (Fig. 6b, d). We confirmed these results through western blot experiments. The MBP protein level decreased in the H/I brains (Fig. 7a, b, p < 0.05). Met treatment significantly increased MBP protein, while this protein expression did not change in the H/I plus vehicle brains (Fig. 7a, b, p < 0.05). We also found that Id2, an inhibitor of MBP expression, changed in the opposite direction of MBP among these four groups (Fig. 7a, b, p < 0.05, respectively).

Fig. 6.

Fig. 6

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Metformin treatment makes OLs increase in the CC. MBP was immunostained at P30. a–d Representative images in the control group (a), H/I group (b), H/I plus Met group (c), H/I plus vehicle group (d), respectively. The intensity of MBP staining was different in the first three groups. Bars, 200 μm

Fig. 7.

Fig. 7

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Metformin promotes MBP and inhibits Id2 expression. Rats were divided into control group, H/I group, H/I plus Met group, and H/I plus vehicle group. After reperfusion, total MBP and Id2 protein were analyzed by western blot. a Immunoblot bands in each group. b The relative bands’ intensity in each group was showed. Data were given as mean ± SD. a p < 0.05, relative to control group. b p < 0.05, relative to H/I group. N = 8 for each group

Metformin Ameliorates Abnormal Myelination in the H/I Rats

We used TEM to detect myelin sheaths and myelinated tracts in the CC. As shown in Fig. 8a, myelin sheaths in the control CC were intact and compact. In the H/I CC, the number of myelin sheaths reduced, and the sheaths were rarefactional. The lamellas were incompact and more irregular. The axoplasms showed shrinkage and the lamellae with disordered structures occasionally separated with the axon (Fig. 8b). After Met treatment, the myelin sheaths increased and became more compact. The axoplasms were more regular. The lamellae and the axons interacted more closely (Fig. 8c). In contrast with these, the vehicle treatment had no effects on the H/I-induced myelination impairments (Fig. 8d).

Fig. 8.

Fig. 8

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Metformin ameliorates abnormal myelination. a–d In the corpus callosum, myelin sheath in each group was characterized through TEM. Myelin sheath decreased and was deformed in the H/I group (b). Met treatment reversed these effects (c), and vehicle addition made no change (d)

Discussion

Hypoxia–ischemia in the preterm neonate can cause the kinds of neurological disorders such as cognitive deficits and motor dysfunction (Carty et al. 2011; Cengiz et al. 2011). Myelination degeneration may be a main reason for the kinds of H/I-induced brain impairments (Qu et al. 2014). Researches on remyelination methods may favor the treatment of related demyelination diseases. Combined the open field test with MWM, our behavioral data revealed that Met treatment ameliorates H/I-induced deficits of locomotor and exploratory activities and has a rescue effect on the spatial memory defects (Figs. 1, 2, 3). The immunostaining and western blot results showed that proliferating OPCs and OLs increase in the Met addition group (Figs. 4, 5, 6, 7). Through TEM analysis, we demonstrated that Met administration rescues the myelin sheath structure injured by H/I (Fig. 8). The vehicle treatment exhibits no significant alteration in H/I-induced brain impairments.

Many brain disorders are due to abnormal neuronal functions and survival. While some researchers focus on the regeneration of neurons, others emphasize the importance of myelination restoration (Zhou et al. 2013). It has been shown that WM impairments are associated with poor neuropsychological performance (Fjell et al. 2011; Nosarti et al. 2004). CC is the largest WM structure in the forebrain. It contains approximately 200 million axons derived from projection neurons in both sides, exhibiting a two-way communication between the two cerebral hemispheres (Epelman et al. 2012). The severe level of cognitive dysfunction is related with the degree of CC injury (Nosarti et al. 2004). OLs are the principal cells in the CC (Follett et al. 2000; Back et al. 2001). H/I can induce glutamate neuron excitotoxicity, inflammation, and free-radical damage. It has been shown that developing OLs are susceptible to these injury forms (Volpe 2005; Jensen 2006). Here we showed that H/I causes defects of spatial learning and memory and locomotor activity in the rats (Figs. 1, 2, 3). Besides, the OPCs and OLs reduce in the H/I group. After Met administration, the behavior activities are improved, and the proliferation of OPCs and the expression of OLs remarkably increase (Figs. 4, 5, 6, 7). These data indicated that Met attenuates the behavioral defects through promoting the generation and maturation of OLs to ameliorate CC impairments.

Myelin sheath, formed by mature OLs, is important for axonal protection and insulation. In addition, myelin sheaths can produce neurotrophic factors which favor neuronal survival (Lin et al. 2004). H/I injury causes defective OLs. The degeneration of OLs leads to the collapse of myelin sheath. Demyelination contributes to the adverse effects on the function and survival of neurons, leading to long-term disorders such as learning defects and mental disorders (Huang et al. 2009). Through electron microscopy analysis, we found that myelin sheaths decrease and are deformed in the H/I CC (Fig. 8b). Met treatment reverses these effects (Fig. 8c), suggesting the inhibition of myelin sheath degeneration.

In conclusion, novel data are revealed that Met promotes OLs’ regeneration and myelin sheath recovery, and ameliorates the impairments of behaviors like spatial learning and locomotor activity in the H/I brains. Because the majority of preterm infants need to be treated after identifying an insult, the observation that Met treatment to H/I rats is protective suggests clinical feasibility. So far, we do not know the mechanisms that Met works. Further studies are awaited to reveal the detailed signaling pathways and clarify the exact key factors. These research progresses may be useful for clinical therapeutic application of Met.

Acknowledgements

This work was supported by the bureau of Xuzhou city science and technology (KC15SH037).

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interests.

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