Orexin-A promotes motor function recovery of rats with spinal cord injury by regulating ionotropic glutamate receptors

doi:10.12122/j.issn.1673-4254.2025.05.15

Abstract

Abstract:

Objective To investigate the effect of orexin-A-mediated regulation of ionotropic glutamate receptors for promoting motor function recovery in rats with spinal cord injury (SCI). Methods Thirty-six newborn SD rats (aged 7-14 days) were randomized into 6 groups (n=6), including a normal control group, a sham-operated group, and 4 SCI groups with daily intrathecal injection of saline, DNQX, orexin-A, or orexin-A+DNQX for 3 consecutive days after PCI. Motor function of the rats were evaluated using blood-brain barrier (BBB) score and inclined plane test 1 day before and at 1, 3, and 7 days after SCI. For patch-clamp experiment, spinal cord slices from newborn rats in the control, sham-operated, SCI, and SCI+orexin groups were prepared, and ventral horn neurons were acutely isolated to determine the reversal potential and dynamic indicators of glutamate receptor-mediated currents under glutamate perfusion. Results At 3 and 7 days after SCI, the orexin-A-treated rats showed significantly higher BBB scores and grip tilt angles than those with other interventions. Compared with those treated with DNQX alone, the rats receiving the combined treatment with orexin and DNQX had significantly higher BBB scores and grip tilt angles on day 7 after PCI. In the patch-clamp experiment, the ventral horn neurons from SCI rat models exhibited obviously higher reversal potential and greater rise slope of glutamate current with shorter decay time than those from sham-operated and orexin-treated rats. Conclusion Orexin-A promotes motor function recovery in rats after SCI possibly by improving the function of the ionotropic glutamate receptors.

Key words: orexin-A, ionotropic glutamate receptor, spinal cord injury, spinal cord ventral horn neurons, patch-clamp recording

Guanglü HE, Wanyu CHU, Yan LI, Xin SHENG, Hao LUO, Aiping XU, Mingjie BIAN, Huanhuan ZHANG, Mengya WANG, Chao ZHENG. Orexin-A promotes motor function recovery of rats with spinal cord injury by regulating ionotropic glutamate receptors[J]. Journal of Southern Medical University, 2025, 45(5): 1023-1030.

Figures/Tables 5

Fig.1 Results of behavioral test of the rats in different groups. A, B: BBB scores in the 6 groups. C: Results of inclined plane test in the 6 groups. D: Comparisons of inclined plane test among SCI+Orexin group, SCI+DNQX group and SCI+Orexin+DNQX group。 aP<0.01, bP<0.01 vs Sham group; cP<0.01 vs SCI+Saline group; dP<0.01, fP<0.01 vs SCI+DNQX group; eP<0.01 vs SCI+Orexin+DNQX group.

Fig.2 A neuron isolated from the ventral horn of the spinal cord captured under high magnification showing clear protrusions from the cell body.

Fig.3 Effect of orexin on reversal potential of glutamate-induced currents in spinal cord ventral horn neurons of rats after spinal cord injury (SCI). A, B: Changes in glutamate-induced currents in normal group under different holding potentials (-70 to +40 mV) and linear fit of the current-voltage relationship curve (the intersection point on the X-axis represents reversal potential of glutamate-induced currents). C, D: Changes in glutamate-induced currents in Sham group under different holding potentials and linear fit of the current-voltage relationship curve. E, F: Changes in glutamate-induced currents in SCI group under different holding potentials and linear fit of the current-voltage relationship curve. G, H: Changes in glutamate-induced currents in SCI+Orexin group under different holding potentials and linear fit of the current-voltage relationship curve.

Fig.4 Comparisons of the reversal potentials of glutamate currents in the spinal cord ventral horn neurons among different groups. **P<0.01.

Fig.5 Comparisons of the dynamic index of glutamate currents in spinal cord ventral horn neurons among the groups. A: Comparison of the rise time of glutamate current. B: Comparison of the rise slope of glutamate current. C: Comparisons of decay time of glutamate current. D: Comparison of decay slope of glutamate current. *P<0.05, **P<0.01.

References 36

1	Wagner FB, Mignardot JB, Le Goff-Mignardot CG, et al. Targeted neurotechnology restores walking in humans with spinal cord injury[J]. Nature, 2018, 563(7729): 65-71.
2	Angeli CA, Boakye M, Morton RA, et al. Recovery of over-ground walking after chronic motor complete spinal cord injury[J]. N Engl J Med, 2018, 379(13): 1244-50.
3	Sakurai T, Amemiya A, Ishii M, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior[J]. Cell, 1998, 92(4): 573-85.
4	Chieffi S, Carotenuto M, Monda V, et al. Orexin system: the key for a healthy life[J]. Front Physiol, 2017, 8: 357.
5	Yamuy J, Fung SJ, Xi MC, et al. State-dependent control of lumbar motoneurons by the hypocretinergic system[J]. Exp Neurol, 2010, 221(2): 335-45.
6	Carpi M, Mercuri NB, Liguori C. Orexin receptor antagonists for the prevention and treatment of Alzheimer's disease and associated sleep disorders[J]. Drugs, 2024, 84(11): 1365-78.
7	Becquet L, Abad C, Leclercq M, et al. Systemic administration of orexin A ameliorates established experimental autoimmune encephalomyelitis by diminishing neuroinflammation[J]. J Neuroinflammation, 2019, 16(1): 64.
8	Hwang YT, Piguet O, Hodges JR, et al. Sleep and orexin: a new paradigm for understanding behavioural-variant frontotemporal dementia[J]? Sleep Med Rev, 2020, 54: 101361.
9	Lee MG, Hassani OK, Jones BE. Discharge of identified orexin/hypocretin neurons across the sleep-waking cycle[J]. J Neurosci, 2005, 25(28): 6716-20.
10	Mileykovskiy BY, Kiyashchenko LI, Siegel JM. Behavioral correlates of activity in identified hypocretin/orexin neurons[J]. Neuron, 2005, 46(5): 787-98.
11	Takakusaki K, Takahashi K, Saitoh K, et al. Orexinergic projections to the cat midbrain mediate alternation of emotional behavioural states from locomotion to cataplexy[J]. J Physiol, 2005, 568(Pt 3): 1003-20.
12	Zhang J, Li B, Yu L, et al. A role for orexin in central vestibular motor control[J]. Neuron, 2011, 69(4): 793-804.
13	Yamuy J, Fung SJ, Xi MC, et al. Hypocretinergic control of spinal cord motoneurons[J]. J Neurosci, 2004, 24(23): 5336-45.
14	Kang JW, Ren BK, Huang LH, et al. Orexin-a alleviates ferroptosis by activating the Nrf2/HO-1 signaling pathway in traumatic brain injury[J]. Aging, 2024, 16(4): 3404-19.
15	Russell Huie J, Stuck ED, Lee KH, et al. AMPA receptor phosphorylation and synaptic colocalization on motor neurons drive maladaptive plasticity below complete spinal cord injury[J]. eNeuro, 2015, 2(5): ENEURO.0091-15.2015.
16	Follwell MJ, Ferguson AV. Cellular mechanisms of orexin actions on paraventricular nucleus neurones in rat hypothalamus[J]. J Physiol, 2002, 545(3): 855-67.
17	Borgland SL, Storm E, Bonci A. Orexin B/hypocretin 2 increases glutamatergic transmission to ventral tegmental area neurons[J]. Eur J Neurosci, 2008, 28(8): 1545-56.
18	Lambe EK, Aghajanian GK. Hypocretin (orexin) induces calcium transients in single spines postsynaptic to identified thalamocortical boutons in prefrontal slice[J]. Neuron, 2003, 40(1): 139-50.
19	Niknia S, Kaeidi A, Hajizadeh MR, et al. Neuroprotective and antihyperalgesic effects of orexin-a in rats with painful diabetic neuropathy[J]. Neuropeptides, 2019, 73: 34-40.
20	Jin N, Zhu SY, Yang XY, et al. Orexin-a potentiates Glycine currents by activating OX₁R and IP₃/Ca²⁺/PKC signaling pathways in spinal cord ventral horn neurons[J]. Brain Res Bull, 2021, 169: 196-204.
21	Thomas A, Miller A, Roughan J, et al. Efficacy of intrathecal morphine in a model of surgical pain in rats[J]. PLoS One, 2016, 11(10): e0163909.
22	Rana S, Sieck GC, Mantilla CB. Diaphragm electromyographic activity following unilateral midcervical contusion injury in rats[J]. J Neurophysiol, 2017, 117(2): 545-55.
23	Fan L, Li XB, Liu T. Asiaticoside inhibits neuronal apoptosis and promotes functional recovery after spinal cord injury in rats[J]. J Mol Neurosci, 2020, 70(12): 1988-96.
24	Anjum A, Yazid MD, Fauzi Daud M, et al. Spinal cord injury: pathophysiology, multimolecular interactions, and underlying recovery mechanisms[J]. Int J Mol Sci, 2020, 21(20): 7533.
25	Reshamwala R, Eindorf T, Shah M, et al. Induction of complete transection-type spinal cord injury in mice[J]. J Vis Exp, 2020(159): (159).
26	Pan LL, Qi RR, Wang JQ, et al. Evidence for a role of orexin/hypocretin system in vestibular lesion-induced locomotor abnormalities in rats[J]. Front Neurosci, 2016, 10: 355.
27	Yan GL, Li FG, Tao ZW, et al. Effects of vestibular damage on the sleep and expression level of orexin in the hypothalamus of rats and its correlation with autophagy and Akt tumor signal pathway[J]. J Oncol, 2022, 2022: 2514555.
28	Iacobucci GJ, Popescu GK. Ca²⁺-dependent inactivation of GluN2A and GluN2B NMDA receptors occurs by a common kinetic mechanism[J]. Biophys J, 2020, 118(4): 798-812.
29	Fani G, Mannini B, Vecchi G, et al. Aβ oligomers dysregulate calcium homeostasis by mechanosensitive activation of AMPA and NMDA receptors[J]. ACS Chem Neurosci, 2021, 12(4): 766-81.
30	Yasuda R, Hayashi Y, Hell JW. CaMKII: a central molecular organizer of synaptic plasticity, learning and memory[J]. Nat Rev Neurosci, 2022, 23(11): 666-82.
31	Kosenkov AM, Gaidin SG, Sergeev AI, et al. Fast changes of NMDA and AMPA receptor activity under acute hyperammonemia in vitro [J]. Neurosci Lett, 2018, 686: 80-6.
32	Plaza-Zabala A, Li X, Milovanovic M, et al. An investigation of interactions between hypocretin/orexin signaling and glutamate receptor surface expression in the rat nucleus accumbens under basal conditions and after cocaine exposure[J]. Neurosci Lett, 2013, 557 Pt B(0 0): 101-6.
33	Dong YJ, Jiang NH, Zhan LH, et al. Soporific effect of modified Suanzaoren Decoction on mice models of insomnia by regulating Orexin-A and HPA axis homeostasis[J]. Biomed Pharmacother, 2021, 143: 112141.
34	Palomba L, Motta A, Imperatore R, et al. Role of 2-arachidonoyl-glycerol and CB1 receptors in orexin-A-mediated prevention of oxygen-glucose deprivation-induced neuronal injury[J]. Cells, 2020, 9(6): 1507.
35	Zhang DX, Cui Y, Zhao MM, et al. Orexin-a exerts neuroprotective effect in experimental intracerebral hemorrhage by suppressing autophagy via OXR1-mediated ERK/mTOR signaling pathway[J]. Front Cell Neurosci, 2022, 16: 1045034.
36	Nepovimova E, Janockova J, Misik J, et al. Orexin supplementation in narcolepsy treatment: a review[J]. Med Res Rev, 2019, 39(3): 961-75.

[1]	Yue CHEN, Linyu XIAO, Lü REN, Xue SONG, Jing LI, Jianguo HU. Monotropein improves motor function of mice with spinal cord injury by inhibiting the PI3K/AKT signaling pathway to suppress neuronal apoptosis [J]. Journal of Southern Medical University, 2025, 45(4): 774-784.
[2]	Linyu XIAO, Ting DUAN, Yongsheng XIA, Yue CHEN, Yang SUN, Yibo XU, Lei XU, Xingzhou YAN, Jianguo HU. Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway [J]. Journal of Southern Medical University, 2024, 44(8): 1589-1598.
[3]	MAN Hao, WANG Jianwei, WU Mao, SHAO Yang, YANG Junfeng, LI Shaoshuo, LÜ Jinye, ZHOU Yue. Jisuikang formula promotes spinal cord injury repair in rats by activating the YAP/PKM2 signaling axis in astrocytes [J]. Journal of Southern Medical University, 2024, 44(4): 636-643.
[4]	SUN Xiaopeng, SHI Hang, ZHANG Lei, LIU Zhong, LI Kewei, QIAN Lingling, ZHU Xingyu, YANG Kangjia, FU Qiang, DING Hua. Exosomes from ectoderm mesenchymal stem cells inhibits lipopolysaccharide-induced microglial M1 polarization and promotes survival of H₂O₂-exposed PC12 cells by suppressing inflammatory response and oxidative stress [J]. Journal of Southern Medical University, 2024, 44(1): 119-128.
[5]	SUN Yang, XU Yibo, XIAO Linyu, ZHU Guoqing, LI Jing, SONG Xue, XU Lei, HU Jianguo. Acetylcorynoline inhibits microglia activation by regulating EGFR/MAPK signaling to promote functional recovery of injured mouse spinal cord [J]. Journal of Southern Medical University, 2023, 43(6): 915-923.
[6]	ZHANG Yu, HONG Shu'e, LIU Jiaming, LIU Zhili, XIAO Shining, YAN Jinxiang, ZHOU Yang. Water tank scale: a reliable method for assessing motor function after spinal cord injury in rats [J]. Journal of Southern Medical University, 2023, 43(1): 99-104.
[7]	. Orexin-A inhibits γ-aminobutyric acid current of neonatal rat spinal cord ventral horn neurons by activating OX1R, OX2R and Ca2+-independent PKC [J]. Journal of Southern Medical University, 2021, 41(5): 694-701.
[8]	LI Xialin, PAN Dayu. Inhibition of TGF-β promotes functional recovery of spinal cord injury in mice by reducing fibronectin deposition [J]. Journal of Southern Medical University, 2021, 41(11): 1686-1691.
[9]	. Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats [J]. Journal of Southern Medical University, 2020, 40(05): 676-682.
[10]	. Icariin alleviates lipid peroxidation after spinal cord injury in rats [J]. Journal of Southern Medical University, 2018, 38(06): 711-.
[11]	. Efficacy of intramedullary and extramedullary decompression and lavage therapy under microscope for treatment of chronic cervical spinal cord injury [J]. Journal of Southern Medical University, 2018, 38(02): 174-.
[12]	. Rebound depolarization of substantia gelatinosa neurons and its modulatory mechanisms in rat spinal dorsal horn [J]. Journal of Southern Medical University, 2017, 37(02): 204-.
[13]	. Effect of suppressing apoptosis signal regulating kinase 1 on GFAP and vimentin expression and hindlimb mobility in rats after spinal cord injury [J]. Journal of Southern Medical University, 2015, 35(06): 795-.
[14]	. Effect of Gold Belt combined with methylprednisolone on motor function and brain-derived neurotrophic factor expression in rats following traumatic spinal cord injury [J]. Journal of Southern Medical University, 2015, 35(02): 276-.
[15]	. Impact of topographic features of electrospun polymethylmethacrylate nanofibers on growth pattern of rat primary astrocytes [J]. Journal of Southern Medical University, 2014, 34(11): 1569-.