Intraperitoneal ozone injection prevents rem sleep deprivation - induced spatial learning and memory deficits by suppressing the expression of Sema3A in the hippocampus in rats

Document Type : Original Article


1 School of Anesthesiology, Weifang Medical University & Department of Anesthesiology, Pain & Sleep Medicine, Affiliated Hospital of Weifang Medical University, Shandong, China

2 Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China

3 College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

4 Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburg 15213, PA, USA

5 Department of Anesthesiology, Pain & Sleep Medicine, Aviation General Hospital of China Medical University & Beijing Institute of Translational Medicine, Chinese Academy of Sciences, Beijing 100012, China

6 Key Laboratory of Mental Health, Peking University Sixth Hospital, Peking University Institute of Mental Health, Ministry of Health (Peking University), National Clinical Research Center for Mental Disorders (Peking University Sixth Hospital), Beijing 100191, China


Objective(s): Sleep deprivation is a common health problem in modern society and is negatively associated with deleterious effects on cognitive functions such as learning and memory ability. This study was undertaken to provide a detailed account of the effect of chronic ozone intraperitoneal injection on the deleterious effects of sleep deprivation on brain function in rats. 
Materials and Methods: Sleep deprivation was induced using the modified multiple platform model. The rats received REM sleep deprivation with an intraperitoneal injection of ozone or midazolam for 28 days. The effects of ozone on REM sleep deprivation-induced hippocampus-dependent learning and memory deficits were studied by the following approaches: Morris water maze (MWM) tests were used to evaluate spatial learning and memory of rats. Morphological changes in the brain were evaluated using hematoxylin and eosin (HE) staining. RNA-sequence was performed to seek a common mechanism. The expression of the targeted gene was examined by qPCR and Western blotting.
Results: Ozone intraperitoneal injection reversed spatial learning and memory deficits associated with REM sleep deprivation, ameliorating pathological brain damage and down-regulating the hippocampal expression of Sema3A in rats after REM sleep deprivation. 
Conclusion: Ozone intraperitoneal injection alleviated sleep deprivation-induced spatial learning and memory deficits by protecting hippocampal neurons via down-regulation of the expression of Sema3A in the hippocampus.


1. Abel T, Havekes R, Saletin JM, Walker MP. Sleep, plasticity and memory from molecules to whole-brain networks. Curr Biol 2013; 23: R774-788.
2. Alzoubi KH, Mayyas F, Abu Zamzam HI. Omega-3 fatty acids protects against chronic sleep-deprivation induced memory impairment. Life Sci 2019; 227: 1-7.
3. Looti Bashiyan M, Nasehi M, Vaseghi S, Khalifeh S. Investigating the effect of crocin on memory deficits induced by total sleep deprivation (TSD) with respect to the BDNF, TrkB and ERK levels in the hippocampus of male Wistar rats. J Psychopharmacol 2021; 35: 744-754.
4. Mhaidat NM, Alzoubi KH, Khabour OF, Tashtoush NH, Banihani SA, Abdul-razzak KK. Exploring the effect of vitamin C on sleep deprivation induced memory impairment. Brain Res Bull 2015; 113: 41-47.
5. Alhaider IA, Aleisa AM, Tran TT, Alzoubi KH, Alkadhi KA. Chronic caffeine treatment prevents sleep deprivation-induced impairment of cognitive function and synaptic plasticity. Sleep 2010; 33: 437-444.
6. Sanday L, Zanin KA, Patti CL, Tufik S, Frussa-Filho R. Role of state-dependency in memory impairment induced by acute administration of midazolam in mice. Prog Neuropsychopharmacol Biol Psychiatry 2012; 37: 1-7.
7. Nielsen S. Benzodiazepines. Curr Top Behav Neurosci 2017; 34: 141-159.
8. Misra AK, Sharma PK. Pharmacotherapy of insomnia and current updates. J Assoc Physicians India 2017; 65: 43-47.
9. Manoto SL, Maepa MJ, Motaung SK. Medical ozone therapy as a potential treatment modality for regeneration of damaged articular cartilage in osteoarthritis. Saudi J Biol Sci 2018; 25: 672-679.
10.    Bocci VA, Zanardi I, Travagli V. Ozone acting on human blood yields a hormetic dose-response relationship. J Transl Med 2011; 9: 66.
11.    Resitoglu B, Celik Y, Komur M, Polat A, Erdogan S, Arslankoylu AE, et al. The efficacy of ozone therapy in neonatal rats with hypoxic ischemic brain injury. Bratisl Lek Listy 2018; 119: 81-85.
12.    El-Mehi AE, Faried MA. Controlled ozone therapy modulates the neurodegenerative changes in the frontal cortex of the aged albino rat. Ann Anat 2020; 227: 151428.
13.    Lin S-Y, Ma J, An J-X, Qian X-Y, Wang Y, Cope DK, et al. Ozone inhibits APP/Aβ production and improves cognition in an APP/PS1 transgenic mouse model. Neuroscience 2019; 418: 110-121.
14.    Li Y, Feng X, Ren H, Huang H, Wang Y, Yu S. Low-dose ozone therapy improves sleep quality in patients with insomnia and coronary heart disease by elevating serum BDNF and GABA. Bull Exp Biol Med 2021; 170: 493-498.
15.    Borrego A, Zamora ZB, González R, Romay C, Menéndez S, Hernández F, et al. Protection by ozone preconditioning is mediated by the anti-oxidant system in cisplatin-induced nephrotoxicity in rats. Mediators Inflamm. 2004; 13: 13-19.
16.    Morsy MD, Hassan WN, Zalat SI. Improvement of renal oxidative stress markers after ozone administration in diabetic nephropathy in rats. Diabetol Metab Syndr. 2010; 2:29.
17.    Radhakrishnan A, Jayakumari N, Kumar VM, Gulia KK. Sleep promoting potential of low dose α-Asarone in rat model. Neuropharmacology. 2017; 125: 13-29.
18.    Alzoubi KH, Mayyas FA, Khabour OF, Bani Salama FM, Alhashimi FH, Mhaidat NM. Chronic melatonin treatment prevents memory impairment induced by chronic sleep deprivation. Mol Neurobiol. 2016; 53: 3439-3447.
19.    Machado RB, Hipólide DC, Benedito-Silva AA, Tufik S. Sleep deprivation induced by the modified multiple platform technique: quantification of sleep loss and recovery. Brain Res 2004; 1004: 45-51.
20.    Datta S, Mavanji V, Ulloor J, Patterson EH. Activation of phasic pontine-wave generator prevents rapid eye movement sleep deprivation-induced learning impairment in the rat: a mechanism for sleep-dependent plasticity. J Neurosci 2004; 24: 1416-1427.
21.    Cao R, Huang F, Wang P, Chen C, Zhu G, Chen L, et al. Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats. Br J Oral Maxillofac Surg 2015; 53: 430-435.
22.    Liang C, Yang T, Wu G, Li J, Geng W. Therapeutic effect of low-intensity pulsed ultrasound on temporomandibular joint injury induced by chronic sleep deprivation in rats. Am J Transl Res. 2019; 11: 3328-3340.
23.    Lu C, Lv J, Dong L, Jiang N, Wang Y, Fan B, et al. The protective effect of 20(S)-protopanaxadiol (PPD) against chronic sleep deprivation (CSD)-induced memory impairments in mice. Brain Res Bull 2018; 137: 249-256.
24.    Jitsuki-Takahashi A, Jitsuki S, Yamashita N, Kawamura M, Abe M, Sakimura K, et al. Activity-induced secretion of semaphorin 3A mediates learning. Eur J Neurosci 2021; 53: 3279-3293.
25.    Yamashita N, Aoki R, Chen S, Jitsuki-Takahashi A, Ohura S, Kamiya H, et al. Voltage-gated calcium and sodium channels mediate Sema3A retrograde signaling that regulates dendritic development. Brain Res 2016; 1631: 127-136.
26.    Banks S, Dinges DF. Behavioral and physiological consequences of sleep restriction. J Clin Sleep Med 2007; 3: 519-528.
27.    Liew SC, Aung T. Sleep deprivation and its association with diseases- a review. Sleep Medicine 2020; 77: 192-204.
28.    Ocalan B, Cakir A, Koc C, Suyen GG, Kahveci N. Uridine treatment prevents REM sleep deprivation-induced learning and memory impairment. Neurosci Res 2019; 148: 42-48.
29.    Zhang W, Zhang W, Dai N, Han C, Wu F, Wang X, et al. A Rat Model of Central Fatigue Using a Modified Multiple Platform Method. J Vis Exp 2018; 138: 57362.
30.    Braidy N, Izadi M, Sureda A, Jonaidi-Jafari N, Banki A, Nabavi SF, et al. Therapeutic relevance of ozone therapy in degenerative diseases: Focus on diabetes and spinal pain. J Cell Physiol 2018; 233: 2705-2714.
31.    Tahmasebi S, Qasim MT, Krivenkova MV, Zekiy AO, Thangavelu L, Aravindhan S, et al. The effects of oxygen-ozone therapy on regulatory T-cell responses in multiple sclerosis patients. Cell Biol Int. 2021; 45: 1498-1509.
32.    Tarkington BK, Duvall TR, Last JA. Ozone exposure of cultured cells and tissues. Methods Enzymol. 1994;234:257-65.
33.    Bocci V, Borrelli E, Travagli V, Zanardi I. The ozone paradox: ozone is a strong oxidant as well as a medical drug. Med Res Rev. 2009;29:646-682.
34.    Bello-Medina PC, Rodríguez-Martínez E, Prado-Alcalá RA, Rivas-Arancibia S. Ozone pollution, oxidative stress, synaptic plasticity, and neurodegeneration. Neurologia (Engl Ed). 2019.
35.    Scassellati C, Galoforo AC, Bonvicini C, Esposito C, Ricevuti G. Ozone: a natural bioactive molecule with anti-oxidant property as potential new strategy in aging and in neurodegenerative disorders. Ageing Res Rev 2020; 63: 101138.
36.    Tirelli U, Cirrito C, Pavanello M, Piasentin C, Lleshi A, Taibi R. Ozone therapy in 65 patients with fibromyalgia: An effective therapy. Eur Rev Med Pharmacol Sci 2019; 23: 1786-1788.
37.    Gupta G, Mansi B. Ozone therapy in periodontics. J Med Life 2012; 5: 59-67.
38.    An JX, Liu H, Chen RW, Wang Y, Zhao WX, Eastwood D, et al. Computed tomography-guided percutaneous ozone injection of the Gasserian ganglion for the treatment of trigeminal neuralgia. J Pain Res 2018; 11: 255-263.
39.    Bocci V, Aldinucci C, Mosci F, Carraro F, Valacchi G. Ozonation of human blood induces a remarkable upregulation of heme oxygenase-1 and heat stress protein-70. Mediators Inflamm. 2007; 2007: 26785.
40.    Kanto JH. Midazolam: the first water-soluble benzodiazepine. Pharmacology, pharmacokinetics and efficacy in insomnia and anesthesia. Pharmacotherapy 1985; 5: 138-155.
41.    Fisher J, Hirshman E, Henthorn T, Arndt J, Passannante A. Midazolam amnesia and short-term/working memory processes. Conscious Cogn 2006; 15: 54-63.
42.    Nyhus E, Curran T. Midazolam-induced amnesia reduces memory for details and affects the ERP correlates of recollection and familiarity. J Cogn Neurosci 2012; 24: 416-427.
43.    Good PF, Alapat D, Hsu A, Chu C, Perl D, Wen XP, et al. A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer’s disease.  J Neurochem 2004; 91:716-736.
44.    Williams A, Piaton G, Aigrot MS, Belhadi A, Theaudin M, Petermann F, et al. Semaphorin 3A and 3F: Key players in myelin repair in multiple sclerosis? Brain 2007; 130: 2554-2565.