Mitochondrial Proteins Unveil The Mechanism By Which Physical Exercise Ameliorates Memory, Learning And Motor Activity in Hypoxic Ischemic Encephalopathy Rat Model Part 3

4. Materials and Methods

4.1. Animals and Experimental Groups

Thirty-six Sprague-Dawley rats were used for this study. The animals were maintained at 22 ± 1 ◦C with light/dark cycles of 12 h and had free access to food and water. Experimental procedures were conducted with the approval of the Ethical Institutional Committee on Animal Care and Research of Zhengzhou University. 

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Every effort was made to reduce the number of animals and minimize animal suffering during the experiments. Animals were randomly divided into four groups (Figure 8). (1) HIE, NT (hypoxia-ischemia encephalopathy group without exercise training) consisted of animals that were modeled for hypoxic-ischemic encephalopathy on postnatal day 7 by anesthetizing the pups with isoflurane (5% induction, 1.5% maintenance). 

The left common carotid artery was then permanently ligated, and the pups were put on a warm recovery couch for 30 minutes before they were transferred to a hypoxic chamber with a continuous flow of the hypoxic gas mixture of 92% N2 and 8% O2 for 90 min and then returned to the cages up to the 10th week without subjecting them to swimming exercise. (2) HIE, T (hypoxia-ischemia encephalopathy group with exercise training) was comprised of animals that were modeled for hypoxic-ischemic encephalopathy as in (1) and returned to the cages up to the 6th week of life and then subjected to 90 min of swimming exercise daily for five days with 2 days of rest per week for 4 weeks, (3) SHAM, NT (control group) included animals that were not subjected to any treatment but lived a normal life in the cages from postnatal day 1 to the 10th week without exercise training, and (4) SHAM, T consisted of animals not subjected to any treatment but lived a normal life in the cages from postnatal day1 up to 6 weeks, after which they were subjected to swimming exercise for 4 weeks.

The animal brains from the different groups were then harvested and prepared for western blotting (hippocampus and cerebral cortex) and immunofluorescence (motor cortex).

4.2. Exercise Paradigm

Animals were housed with food and water ad libitum and maintained on a 12-light/dark cycle. They were divided into two groups: sedentary animals (HIE, NT and SHAM, NT) and exercising animals (HIE, T and SHAM, T). The exercising animals were subjected to swimming exercise training after postnatal week 6 for 4 weeks; they were subjected to swimming exercise for 5 days a week with 2 days of rest. Each swimming session lasted for 90 min. The pool was filled to a depth of 50 cm to prevent the animals from touching the bottom of the tank. 

The animals were allowed to freely swim, without any extra load, and were gently stimulated during swimming. The rats were carefully dried after the exercise and returned to the cages.

4.3. Vertica! Pole Motor Test

The animals in each group were one by one placed face-up on a cloth tape-covered pole (3.0 cm diameter and 150 cm length), which was held in a horizontal position, then was gradually lifted to a vertical position, and the time the rat stayed on the pole was recorded for a maximum of 2 min (120 s). In this test, the animal with a deficit in motor coordination and balance falls off the pole.

4.4. Morris Water Maze Test (MWM)

Rats (10 weeks old) were subjected to MWM after undergoing 4 weeks of exercise training protocol (SHAM, 'T and HlE, T). All water maze data were recorded using the Panjab SMART video tracking system (Panlab Howo Biotechnology (Shanghai) Co., Ltd.)(Shanghai, China). The MWM was used as described by l32]. Briefly, rats used visual cues placed on the borders of a swimming pool to reach a hidden platform and escape from the water. Learning was assessed across 7 days. Before the learning assessment, rats were introduced into the pool that contained clear water and a visible platform. During the training, the rats were given trial swimming exercises to become familiar with the task.

During the learning phase, rats were smeared with black dye for easy video tracking and the platform was submerged. Each rat was subjected to four trials from different starting points (quadrants). Latency or the time required to reach the platform was recorded every day by the Panlab SMART Video Tracking System. On the last day of the experiment, the platform was removed, and each rat was reintroduced into the water the number of times the animals crossed the site of the hidden platform in the quadrant that previously contained the platform (target quadrant) was recorded.

4.5. Western Blotting

Six rats were randomly selected from each group for the protein-level experiment. Proteins were extracted from the hippocampus and cortex and homogenized in RIPA's reagent (CW2333S, CoWin Biosciences, Cambridge, MA, USA) and separated into cytoplasmic and nuclear proteins. The protein concentrations were determined using the bicinchoninic acid assay (BCA) (CW0014S, CoWin Biosciences, Cambridge, MA, USA), and a total of 20 g of protein was separated by electrophoresis on universal SDS-PAGE gels (CFAS Any KD PAGE) # PE008, Zhonghui Hecai Bio-pharmaceutical Technology Co., Ltd., Shaanxi, China. 

Proteins were then transferred onto polyvinylidene fluoride membranes, (PVDF) (R1CB12934, Merck Millipore Ltd., (Burlington, MA, USA). The membranes were blocked for 2 h with 5% bovine serum albumin (BSA) (#A8020, Solarbio Life Sciences, Beijing, China) at room temperature and incubated with the following primary antibodies from Cell Signaling Technology (Danvers, MA, USA): AIF (D39D2, 1:1000), Cytochrome C (136F3, 1:1000), cleaved caspase-3 (Asp175, 1:1000), Smac/Diablo (D553R, 1:1000), and OPA1 (D7C1A, 1:1000). 

GAPDH from Servicebio, Wuhan, China (GB11002, 1:2000), and H3 from Proteintech (17168-1-AP, 1:1000) at 4 ◦C overnight followed by HRP-conjugated secondary antibody SA00001-2 from Proteintect (1:5000) incubation for 2 h at room temperature. The protein bands were visualized with an enhanced chemiluminescence kit #KF005 from Affinity Biosciences and imaged with the Bio-Image Analysis system (Zhengzhou University, Zhengzhou, China). The ratios of protein band intensities to GAPDH (cytoplasmic proteins) and H3 (nuclear proteins) as internal references were determined using ImageJ.

4.6. Immunofluorescence

The 25-µm coronal slices of brain tissue were obtained using a freezing microtome (Leica, Germany) for immunofluorescence. The slides were blocked for 2 h with PBS with 10% fetal bovine serum, (#A8020, Solarbio Life Sciences, Beijing, China) and 0.3% Triton ×-100, Amresco 0694, Biosharp, Estonia at room temperature and then incubated with primary antibodies from Cell Signaling Technology (Danvers, MA, USA): anti-AIF, D39D2 (dilution of 1:400), anti-Cytochrome C, 136F3 (dilution of 1:200), anti-cleaved caspase-3, Asp175 (dilution of 1:400), anti-Smac/Diablo, D553R (dilution of 1:200) and anti- OPA1, D7C1A (dilution of 1:400) overnight at 4 ◦C. 

Then, the slides were washed 3 times each lasting 10 min in PBS, and then incubated with secondary antibodies (ab150077 from Abcam, Cambridge Biomedical Campus, Cambridge, UK) for 2 h at room temperature in the dark. DAPI,#C0065 from Solarbio Life Sciences, Beijing, China (1:100PBS) was added for 5 min and poured off, washed in PBS for 3 times each lasting 5 min, dried, the anti-quenching agent was added(#P0126, Beyotime Biotechnology, Shanghai, China), glass slips were fixed on the slides, and samples were stored at −20 ◦C for 2 h and taken for microscopic imaging. The tissue images were captured using a confocal fluorescence microscope (Ni-U942877, Nikon, Tokyo, Japan), and the images representing the proteins in the motor cortex were then quantified using FIJI-ImageJ as described in [33].

4.7. Statistical Analysis

GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, CA, USA) was used for all analyses. One-way ANOVA followed by Dunnett's post hoc tests, respectively, were used to measure statistical significance. Results are presented as mean ± SEM, and p < 0.05 was considered statistically significant.

5. Conclusions

This study demonstrates that swimming exercise ameliorated motor activity, memory, and learning associated with HIE by suppressing mitochondrial apoptosis via the Cyto.C/cleaved caspase-3 and AIF signaling pathways. Additionally, swimming exercise intervention has been shown to stabilize the mitochondrial cristae and membrane potential as depicted by the reversal of Smac/Diablo and OPA1 in rats with HIE. As a result, this is an attractive and viable foundation for additional research to decipher more molecular pathways that could be manipulated to effectively manage HIE and the resultant deficits. While this study successfully demonstrated the role of exercise in reversing the structural and functional deficits in the mitochondria caused by HIE, it did not exhaust all the possible methods for establishing how the mitochondrial cristae are stabilized; this is critical for future research on this subject.

Author Contributions: Conceptualization, F.G., F.P., Y.W., and C.C.; data curation, F.G., F.P., Y.W., H.L., and J.F.; formal analysis, F.G., H.L., and C.C.; funding acquisition, C.C.; investigation, F.G., F.P., Y.W., H.L., and J.F.; methodology, F.G., F.P., Y.W., and C.C.; project administration, F.G., Y.W., and C.C.; resources, C.C.; software, F.G., F.P., J.F., and C.C.; supervision, C.C.; validation, F.G., F.P., Y.W., H.L., and C.C; visualization, F.G., F.P., Y.W., H.L., and J.F.; writing–original draft, F.G., and Y.W.; writing–review and editing F.G. and H.L. All authors have read and agreed to the published version of the manuscript.

Funding: This study is supported by the Scientific and Technological Research Project of the Henan Provincial Department of Science and Technology (Grant No. 212102310217) and a grant from the Funds of the National Natural Science Foundation of China (Grant No. 81401015).

Institutional Review Board Statement: The study was conducted according to the guidelines of the Committee for the Care of Research Animals of Zhengzhou University; ZZUIRB2022-32.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Conflicts of Interest: The authors declare no conflict of interest.

References

1. Li, B.; Concepcion, K.; Meng, X.; Zhang, L. Brain-immune interactions in perinatal hypoxic-ischemic brain injury. Prog. Neurobiol. 2017, 159, 50–68. [CrossRef] 

2. Douglas-Escobar, M.; Weiss, M.D. Hypoxic-ischemic encephalopathy: A review for the clinician. JAMA Pediatr. 2015, 169, 397–403. [CrossRef] [PubMed] 

3. Black, R.E.; Cousens, S.; Johnson, H.L.; Lawn, J.; Rudan, I.; Bassani, D.; Jha, P.; Campbell, H.; Walker, C.F.; Cibulskis, R.; et al. Global, regional, and national causes of child mortality in 2008: A systematic analysis. Lancet 2010, 375, 1969–1987. [CrossRef] 

4. Juul, S.E.; Ferriero, D.M. Pharmacologic Neuroprotective Strategies in Neonatal Brain Injury. Clin. Perinatol. 2014, 41, 119–131. [CrossRef]

5. Zhu, C.; Rodriguez, J.I.; Li, T.; Xu, Y.; Sun, Y. Role of apoptosis-inducing factor in perinatal hypoxic-ischemic brain injury. Neural Regen. Res. 2021, 16, 205–213. [CrossRef] [PubMed] 

6. Song, S.-H.; Jee, Y.-S.; Ko, I.-G.; Lee, S.-W.; Sim, Y.-J.; Kim, D.-Y.; Lee, S.-J.; Cho, Y.S. Treadmill exercise and wheel exercise improve motor function by suppressing apoptotic neuronal cell death in brain inflammation rats. J. Exerc. Rehabil. 2018, 14, 911–919. [CrossRef] 

7. Kelly, M.; Darrah, J. Aquatic exercise for children with cerebral palsy. Dev. Med. Child. Neurol. 2005, 47, 838–842. [CrossRef] 

8. Müller, P.; Duderstadt, Y.; Lessmann, V.; Müller, N.G. Lactate and BDNF: Key Mediators of Exercise Induced Neuroplasticity? J. Clin. Med. 2020, 9, 1136. [CrossRef] 

9. Intlekofer, K.A.; Cotman, C.W. Exercise counteracts declining hippocampal function in aging and Alzheimer's disease. Neurobiol. Dis. 2013; in press. [CrossRef] 

10. Real, C.; Ferreira, A.; Chaves-Kirsten, G.; Torrão, A.; Pires, R.; Britto, L. BDNF receptor blockade hinders the beneficial effects of exercise in a rat model of Parkinson's disease. Neuroscience 2013, 237, 118–129. [CrossRef]

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