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Effect of Low Intensity Magnetic Field Stimulation on Calcium-Mediated Cytotoxicity After Mild Spinal Cord Contusion Injury in Rats

Effect of Low Intensity Magnetic Field Stimulation on Calcium-Mediated Cytotoxicity After Mild... Background: Magnetic field (MF) stimulation has the potential to reduce secondary damage and promote functional recovery after neural tissue injury. The study aimed to observe the effect of very low intensity (17.96µT) MF on general body condition, secondary damage, pain status, and locomotion. Methods: We exposed rats to MF (2 h/day × 3 weeks) after 6.25 mm contusion spinal injury. Locomotor behavior was evaluated by BBB score, pain assessment was done by recording threshold for tail flick, expression of voltage-gated calcium channels and extent of secondary damage in the spinal cord was assessed by immunofluorescence and Cresyl violet staining, respectively. Results: A significant (p ≤ .001) improvement in bladder function as well as BBB score was observed after MF exposure in comparison with sham and SCI over the observation period of 3 weeks. SCI group showed an increase in the threshold for vocalization after discharge, which decreased following MF exposure. Cresyl violet staining showed significantly higher tissue sparing (73%) at the epicenter after MF exposure when compared to SCI group. This was accompanied with a significant decrease in calcium channel expression in MF group as compared to SCI. Conclusion: The results suggest facilitation of sensory-motor recovery after MF exposure, which could be due to attenuation of secondary damage and calcium-mediated excitotoxicity in a mild contusion rat model of SCI. Keywords Spinal cord injury, contusion, magnetic field stimulation, locomotion, calcium, pain Abbreviations BBB: Basso, Beattie, and Bresnahan BMR: basal metabolic rate Ca: calcium ion CpG: central pattern generator DAPI: 4,6-diamidino-2-phenylindole FITC: fluorescein isothiocyanate K: potassium ion mA: milliampere MF: magnetic field Na: sodium ion PBS: phosphate buffer saline PBSTx: phosphate buffer saline triton SCI: spinal cord injury TTF: tail flick threshold VAD: vocalization after discharge VDCCs: voltage-dependent calcium channels µT: micro Tesla Faculty of Biological Sciences, University of Leeds, Woodhouse, Leeds, UK. Introduction Institute of Microbial Technology, Chandigarh, India Department of Physiology, All India Institute of Medical Sciences, New Delhi, India Spinal cord injury (SCI) initiates cascade of events, which Corresponding author: include significant electrolytic shifts, involving monovalent Suman Jain, Department of Physiology, All India Institute of Medical Sciences, + + 2+ (Na , K ) and divalent (Ca ) cations imbalance, that Ansari Nagar, New Delhi 110029, India. contributes to excitoxicity, oxidative stress, and degeneration E-mail: sumanjain10@gmail.com Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution- NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-Commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 50 Annals of Neurosciences 27(2) of neuronal tissue due to secondary injury progression. This MF Exposure Chamber leads to loss of sensory, motor, and autonomic functions in The MF exposure chamber (modified form of Helmholtz coil) body parts below the level of lesion. It also results in intense used has 18 and 8 turns of wire wound on circular formers chronic or neuropathic pain due to central sensitization. of two outer and inner electromagnetic coils, respectively. Ionic imbalance, specifically mitochondrial calcium overload Rats were kept in a polypropylene cage (50 × 20 × 15 cm3) immediately after SCI, seems to be one of the key players for on central area of the chamber that provides uniform MF of the neuronal death and glutamate-mediated neurotoxicity. 6,13 17.96µT, 50Hz. Transcranial as well as external magnetic field (EMF) stimulation has been shown to improve locomotor deficits, osteoporosis, tonic pain, general body conditions, lesion Procedure for Inducing Spinal Cord Injury 4–8 volume, density of serotonergic fibers at injury site, and Under anesthesia (thiosol; 50 mg/kg body weight, i.p.), attenuate inflammation and microglial activity in complete the dorsal surface of thoracic area was shaved and cleaned transection, hemisection, and compression rat models with povidone-iodine. An incision was made from the upper of SCI. A weight drop contusion rat model is one of the thoracic region to the upper lumbar region. Fascia and muscles most reproducible and reliable animal model for studying were removed from T11–T13 vertebral level (laminectomy) effectiveness of any therapeutic strategy in SCI as it closely to expose spinal T13 level of the cord (sham group). In SCI 10,11 mimics human injuries. Exposure of severely contused SCI and SCI + MF groups after laminectomy, mild contusion rats to pulsed EMF facilitates functional recovery by reducing injury (height of 6.25 mm) was done by New York University oxidative stress, inflammation, and increasing expression of (NYU) SCI contusion device. The body temperature was HSP70. However, the effect of MF on calcium-mediated maintained during surgery by controlled heating pads. cytotoxicity is not clear. Thus, objective of the study was to Suturing of muscle, fascia, and skin was done, and rats were observe the low intensity MF stimulation effect on general kept back in their home cages. To compensate for blood loss, body condition, locomotion, pain, and calcium imbalance in ringer lactate solution (5 mL, i.p.) was provided to rats along mild contusion SCI rats. with antibiotics (50 mg/kg b.wt Gentamycin, im; neosporin ointment applied locally) after surgery. We manually Methods expressed bladder 3 times/day until bladder control restored. The day of return of bladder function to normal was noted for Adult female Wistar rats weighing between 200 and 250 grams each rat of all the groups. were used for the study. Animals were obtained from Central Animal Facility (AIIMS, New Delhi, India) and kept in a room Assessment of Locomotor Functions having controlled temperature (25 ± 2 °C) and light: dark cycle (14:10 h). Ad libitum water and food pellets (Ashirwad The rat was placed in open field for 4 minutes to assess hind Industries Ropar, India) were provided. Committee for the limb movements using Basso, Beattie, and Bresnahan scoring Purpose of Control and Supervision of Experiments on Animals scale, which is based on movement of three hind limb joints (CPCSEA, Animal Welfare Division, Ministry of Environment, (hip, knee, and ankle). Video recording of the locomotor Forest and Climate Change, India) and Institutional Animal behavior of rat was done to perform off-line BBB scoring Ethics Committee (ethics no 966/IAEC/16) approved the between 0 and 21 scale by a blind observer. study. It was a 21 day protocol and after recording all the baseline behavioral recording, the animals were divided into Assessment of Sensory Functions three groups randomly: Sham (n = 6), only laminectomy was performed after anesthesia (Pentabaritol 50 mg/kg b.wt); Threshold for tail flick is a segmental response, which is SCI (n = 6): laminectomy was followed by contusion SCI; mediated via spinal cord. TTF was determined by applying AC SCI + MF (magnetic field exposure) (n = 6): one day after the current to tail of the rat. The rat was habituated in a Plexiglas contusion injury of spinal cord, the rats were exposed to MF restrainer for 30 minutes, before starting the experiment. Two (EMF, 50 Hz, 17.96µT, 2 h/d for 3 weeks). After 21 days of needle electrodes (0.5″ × 27G) were inserted intradermally, injury, all the rats were sacrificed (euthanatized) by transcardial approximately 2 mm deep and 2 cm apart into the middle perfusion under deep anesthesia. portion of the tail (rostrally and caudally) and were fastened Food intake, water intake, body weight, and BBB scoring with adhesive tape. In this test, noxious electrical stimulation were recorded on the 1st, 5th, 7th, 9th, 13th, and 21st day of was applied to stimulate nociceptive afferents (biphasic the study. The threshold for tail flick and vocalization after square wave pulse with frequency 40 Hz, 1.5 ms pulse discharge was quantified on 21st day. SCI + MF group of rats width and varying strength of current from 0.1 to 10 mA were kept in a polypropylene cages and placed in MF chamber (AD Instruments and Lab Chart 8 [MLS060]). The current for 2 h/day for 21 days. After transcardial perfusion, all the at which tail flick occurred was considered as the threshold spinal cord tissues were stored for histological analysis. for tail flick. Bhattacharyya et al. 51 16 rats (sham = 6, SCI = 5, SCI + MF = 4) survived the total Vocalization After Discharge study period of 21 days. The behavioral and histological data Electrical stimulus strength was increased beyond tail flick of these rats were used for analysis and are presented in the threshold in the same rat. A loud vocal sound of higher pitch results. that outlasted the duration of stimulus was noted as VAD. It There was no significant difference in baseline represents emotional component of pain, which is organized presurgery food intake of all the groups, whereas in limbic structure. following SCI food intake decreased significantly in all the groups (sham, p = .001; SCI, p = .001; SCI + MF, p = .033). Food intake was nadir immediately after surgery Histology and gradually increased over the days. In SCI group this After three weeks of surgery, the rats were deeply decrease was maintained till day 5 (p = .039), whereas in anesthetized by injecting thiosol (i.p., 50 mg/kg of body SCI + MF group, no statistically significant difference in weight) and perfused transcardially with cold saline (4 °C) food intake on any of the postoperative days was observed and 4% paraformaldehyde. The spinal cord along with when compared with basal. Intergroup analysis revealed the vertebral column was isolated from T5 to L5 and post a significant decrease in food intake in SCI (p = .02) and fixed with 4% paraformaldehyde at 4 °C. A 6 mm long SCI + MF (p = .03) groups as compared to sham (Figure spinal cord tissue containing lesion epicenter was taken, 1) on day 21 and significant decrease in SCI (p = .001) cryosectioned (16µm thick), and mounted on poly-L-lysine as compared to SCI + MF on day 5. Body weight did not coated slides. Cresyl violet staining of the sections was done change significantly among all the groups after surgery using standard protocol to assess the lesion area extent and (Figure 2). volume. The total area of injury in one section was tracked by light microscope and calculated using NIS-Element Figure 1. Food Intake (gm, Mean ± SD) of All the Rats of All Groups software (Nikon). Immunohistochemistry for L-Type Calcium Channels Sections were collected on poly-L-lysine coated slides and washed with PBSTx three times each for 5 minutes. After that for 2 hours, 10% normal goat serum was added on to the sections for blocking. Then, they were incubated in primary antibody (anti-L-type calcium channel antibody C1353 at concentration 1:200) overnight at 4°C. It was again washed with PBSTx five times each for 3 minutes and then incubated with secondary antibody (fluorescein isothiocyanate conjugated at concentration of 1:400) for 2 hours at room Surce: Authors own. temperature. Finally washing with PBSTx five times for 3 Note: # indicates sham vs SCI/SCI + MF; Food intake increased significantly minutes was done. Slides were mounted in 4,6-diamidino-2- after MF stimulation. phenylindole (DAPI) containing mounting media. Figure 2. Body Weight (Mean ± SD) of All Groups at Different Time Points Statistical Analysis Statistical analysis was performed using Statistical Package for Social Sciences version 15 software (SPSS Inc, Chicago, IL, USA). The data are presented as the mean ± standard deviation. Data of each time point between the groups was compared by the Kruskal–Wallis test with post hoc analysis done by using Dunn’s test with the Bonferroni correction. Statistical significance was accepted at p ≤ .05. Results General Body Condition A total of 18 rats were taken and after surgery, randomly Source: Authors own. divided into three groups (n = 6 rats in each group). A total of Note: No significant change was observed throughout the study period . 52 Annals of Neurosciences 27(2) Figure 3. Day of Restoration of Urinary Bladder Function (Mean Figure 4. Effect of MF Exposure on BBB Score (Mean ± SD) in All ± SD) the Rats at All the Time Points Source: Authors own. Note: # indicates sham vs SCI/ SCI + MF; * indicates SCI vs SCI + MF. A significant improvement in BBB score was observed following MF exposure. Figure 5. Effect of MF Exposure on Threshold for Tail Flick and Vocalization After Discharge (mA, Mean ± SD) Source: Authors own. Note: # indicates sham vs SCI/SCI + MF; * indicates SCI vs SCI + MF. Spontaneous evacuation of bladder was achieved significantly earlier in SCI + MF group. After SCI, there was loss of urinary bladder function in both SCI and SCI + MF groups. Manual evacuation was done eight hourly to survive the rats. A significant effect of intervention was observed on the day of restoration of Source: Authors own. spontaneous bladder function (χ = 12.54; p = .001). In SCI Note: No significant change observed in phasic pain after mild SCI and MF group, post hoc analysis showed that the day of spontaneous exposure. evacuation of bladder was achieved significantly later (day 6.2 ± 1.48, p = .0001) in comparison to sham (day 1.6 ± 0.5). MF exposure had significant effect and in SCI + MF group, Pain Threshold bladder function recovered significantly (day 3.25 ± 0.5; The sensory as well as emotional components of pain were p = .0002) earlier in comparison to SCI group (Figure 3). assessed by recording threshold for tail flick and vocalization after discharge. There was no significant (χ = 4.05, p = Locomotor BBB Score .13) difference in the threshold for tail flick among all the three groups (Figure 5). However, on post hoc analysis, on Bilateral hind limb paralysis occurred in all the rats of all the day 21, a significant increase in SCI + MF group (p = .03) groups after spinal cord contusion injury at thoracic level. was observed as compared to sham. When vocalization after BBB score was significantly different (p ≤ .001) among all discharge threshold was compared between groups, there was the groups following surgery. In SCI + MF and SCI groups, a trend for an increase of the threshold in SCI group and a a significant drop of BBB score was observed after injury decrease in MF exposure group, but it was not statistically from the basal as well as in comparison to sham group. significant ( χ = 2.65, p = .26). Over the observation period of 3 weeks, the score increased gradually in both the groups but was maintained at a lower level (p ≤ .001) in comparison with sham. MF stimulation Histology and Immunohistochemistry significantly improved hind limb function starting from day Morphological analysis of the spinal cord revealed presence 1 of surgery. In MF group, the BBB score was significantly of lesion in the center and sparing at the periphery of the tissue. higher (p ≤ .001) at all time points during 3-week study In Cresyl violet staining, the central lesion area depicted period as compared to SCI group, suggesting beneficial effect cyst formations, gliosis, zones of partial demyelination, of intervention (Figure 4). Bhattacharyya et al. 53 microcysts, scarring, and was mostly localized to dorsal horn. Discussion In SCI group, 51% fibers were spared, whereas following intervention of MF exposure in SCI + MF group, it increased SCI leads to intolerable chronic pain besides severe deficits in 15,16 to 73%. A statistically significant (χ = 8.43, p = .004) motor, autonomic, and sensory functions. Chronic pain is difference in total damage area of spinal cord was observed an unavoidable part of secondary cascade, which ensues after when comparison was done between SCI + MF and spinal cord injury subsidies and is resistant to analgesics. This SCI groups. A significant decrease in the damaged area gives a reason to find an intervention that could effectively following MF exposure for 3 weeks was evident (Figure treat chronic pain. In the recent years, magnetic field has 6). Immunohistochemistry for L-type voltage-dependent shown to improve sensorimotor function, tonic pain and calcium channels (VDCCs) in spinal cord tissue sections was hyperalgesia in complete transection/hemisection models of 4,5,17 performed to assess role of calcium in excitotoxicity. DAPI SCI. The current study was designed to understand low staining revealed calcium localization adjacent to the nucleus, intensity electromagnetic stimulation effect on locomotion, on the cell membrane. A significantly higher expression of chronic pain, and on calcium signaling in mild contusion rat L-type VDCCs in SCI group was evident in comparison to model of SCI. We used a mild thoracic contusion injury model sham. However, we observed that in the SCI + MF group, the in our study as it mimics best the human condition among expression decreased in comparison to SCI (Figure 7). all the other SCI models available. SCI is generally followed by a cascade of events including generation of free radical, Figure 6. (A) Representative Images of Cresyl Violet Staining of ionic imbalance and alteration of cellular events which lead to Spinal Cord in Each Group. (B) Effect of MF exposure on Mean degeneration of cord both rostrally and caudally and thereby (±SD) Damaged and Spared Area in All the Rats. disruption in sensory and motor function. We also observed bilateral hind limb paralysis and loss of bladder function (A) immediately after contusion at T13 in all the rats. Food intake was significantly decreased in spinal cord (B) injured rats. Consistent decrease in food intake was observed till fifth day of injury in SCI group, following which it recovered spontaneously. Since SCI is a type of neurotrauma, thereby a stressful event, initial decrease in feeding behavior in our study was expected. The decrease in body weight and feeding behavior may also be secondary to alterations in gastric 18,19 motility and emptying. During the initial weeks after SCI, even the patients have been shown to have a tendency to lose weight due to muscle atrophy and bone mineral loss resulting in fat free mass loss and lower body mass index. Reduced fat-free mass, sympathetic blunting, cardiopulmonary Note: * indicates SCI vs SCI + MF. A significant decrease in damaged tissue area and an increase in spared tissue area is observed after MF exposure. dysfunction, work capacity reductions, and diminished anabolic hormones also sustain the BMR decrement after the Figure 7. Effect of magnetic field stimulation on voltage-gated cal- acute phase of SCI. In SCI rats, urinary bladder function cium channel expression in spinal cord tissue. The expression was remained disrupted for significantly higher time as compared decreased in MF group as compared to SCI. to sham in the present study. Animal studies have exhibited that reflex micturition is mediated by a spinobulbospinal pathway, whereas urethral relaxation is brought about by excitation of the pontine micturition center, which in turn activates descending pathways. This relaxation eventually is followed by an activation of sacral parasympathetic outflow (L6–S1 in rats), which leads to contraction of the bladder, an increase in intravesical pressure and the flow of urine. Thus, SCI at the thoracic level eliminates voluntary and supraspinal control of voiding, leading to an initial phase of areflexic bladder and complete urinary retention. Finally, there is a slow development of automatic micturition and 23,24 bladder hyperactivity mediated by spinal reflex pathways. Thoracic SCI also can cause complete paralysis or motor deficits in the hind limbs depending on the severity of the injury. Paraplegia was observed immediately following mild thoracic injury in the present study, which gradually partially 54 Annals of Neurosciences 27(2) recovered over the observation period of 21 days postinjury, Voltage-gated calcium channels mediate calcium influx into though it was significantly less than sham group, as assessed living cells upon membrane depolarization and have a critical by BBB score. This gradual recovery in locomotor function is role in calcium signaling. Increase in alpha-2-delta subunit possibly because of the sparing of central pattern generators, of voltage sensitive calcium channel in SCI rats has been which is located at L1–L2 segment in rat, and we gave shown to correlate with development of tactile allodynia of lesion at T13 spinal segment. Further, 51% tissue was spared hind paw. MF stimulation (17.96µT, 50 Hz, 2hrs/day for 3 in the SCI rats, sprouting from them may have contributed weeks) of spinal cord of injured rats, in the present study, to the spontaneous recovery of BBB score. Spared area after reduced the calcium channel expression. In literature, there SCI is a prominent marker for tissue integrity and behavioral are contradictory reports on the effects of EMF on calcium recovery. channels. Low frequency-EMF, 1–25 Hz for <180 ms, with Chronic pain is among the most prominent characteristic 1–2 T intensity reduced the calcium transit from endoplasmic feature of SCI; however, mild injury to the cord could not reticulum in F-11 cells. Sun et al. (2016) reported enhanced alter pain parameters significantly in our study. Spinothalamic calcium channel expression of all the subtypes at presynaptic pathway, which is mainly located in the ventral spinal cord, terminal in situ brain slice preparation, thereby enhancing is majorly responsible for the pain perception. In mild SCI, vesicle endocytosis and synaptic plasticity following there is major damage to dorsal part, mainly consisting of exposure to low frequency MF (uniform 1 mT MF with 50 Hz gracile fasciculus and cuneate fasciculus. These are the first- frequency) for 8–10 days. Low frequency electromagnetic order sensory neurons that relay proprioceptive, fine touch, field (1 mT, 50 Hz) stimulation also enhanced neurogenesis and vibration sensations to the thalamus, thereby the pain via expression of calcium receptor (Ca 1). It is therefore sensation remains unaffected. Under Cresyl violet staining suggested that the expression of calcium channels following of the lesioned spinal cord tissue, we also observed major MF stimulation may depend upon the model used (in vivo or damage to the both fasciculi and sparing of dorsal horn as in vitro), magnetic stimulation parameters, and the design of well as ventral and lateral parts of the spinal cord. the study protocol. MF exposure for 5–8 weeks has been shown to ameliorate locomotor deficits, osteoporosis, tonic pain, and general body Conclusion conditions in completely transected or hemisected spinal cord 5,6,9,17 rat models. MF exposure, for 3 weeks to rats with mild The present study shows beneficial role of short-term MF spinal contusion injury, was able to improve general body exposure in the contusion SCI rat model on locomotor condition like food intake and urinary bladder function. This behavior by preventing calcium-mediated excitotoxicity. is in agreement with previous literature, wherein MF exposure has been shown to improve general body condition and Acknowledgments facilitate early recovery of urinary bladder function. There We would like to thank Mr Pursottam Samal for histology and was also a significant improvement in BBB locomotor scale Department of Biotechnology, AIIMS, for all the support during as compared to SCI, though it was lower than sham group. this research work. We acknowledge the support rendered by Dr This may be due to 73% sparing of neural tissue, which under Anushree Gupta, Associate Professor, AIIMS, Delhi. the influence of magnetic fields was able to regenerate and compensate for the lost tissue. MF exposure has been shown Author Contributions to activate regenerative processes under both in vitro and in 29,30 vivo conditions. It also has proved beneficial in restoring S. J. Conceived the project and majority of the laboratory work has been performed in her laboratory. locomotion, muscle contraction properties, and limiting S. B. and S. S. Performed all the experimental works. muscle degeneration in SCI rats. In total, 5%–10% fibers at S. B. Wrote the manuscript also. the epicenter of contusion (T7–9) and clip compression (T1) S. K. Standardized and helped in performing behavioral experiments. injuries of spinal cord and 1%–5% descending axons have been shown to be sufficient to restore the segmental local Declaration of Conflicting Interests 31,32 circuits necessary for locomotion. After SCI due to ionic imbalance and excitotoxicity of The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. glutamate receptor, calcium concentrations reach critical level, which leads to neuronal loss. We studied L-type Ethical Statement VDCCs, which is a marker of excitotoxicity in neuronal cells to unravel the underlying cellular mechanism of the The study was approved by the Committee for the Purpose of Control beneficial effects of MF. A significantly higher expression was and Supervision on Experiments on Animals (CPCSEA), New observed in SCI as compared to sham and SCI + MF groups. Delhi, Animal Welfare Division under Ministry of Environment, High levels of glutamate cause excitotoxicity by facilitating Forest and Climate Change (Govt. of India) and approved by the 2+ 33 Institutional Animal Ethics Committee (Ethics no 966/IAEC /16). entry of high levels of calcium ions (Ca ) to enter the cell. Bhattacharyya et al. 55 Funding 13. Kirschvink JL. Uniform magnetic fields and double- wrapped coil systems: improved techniques for the design The authors received no financial support for the research, of bioelectromagnetic experiments. Bioelectromagnetics authorship, and/or publication of this article. 1992; 13(5): 401–411. 14. Mukherjee K, Mathur R, and Nayar U. Ventromedial Reference hypothalamic mediation of sucrose feeding induced pain 1. Hulsebosch CE. Recent advances in pathophysiology and modulation. Pharmacol Biochem Behav 2001 Jan; 68(1): 43–48. treatment of spinal cord injury. Adv Physiol Educ 2002 15. Morgan D, Carter CS, DuPree JP, et al. Evaluation of Dec; 26(1–4): 238–255. prescription opioids using operant-based pain measures in rats. 2. Lindsey AE, LoVerso RL, Tovar CA, et al. An analysis of Exp Clin Psychopharmacol 2008 Oct; 16(5): 367–375. changes in sensory thresholds to mild tactile and cold stimuli 16. Gwak YS, Kang J, Unabia GC, et al. Spatial and temporal after experimental spinal cord injury in the rat. Neurorehabil activation of spinal glial cells: role of gliopathy in central Neural Repair 2000; 14(4): 287–300. neuropathic pain following spinal cord injury in rats. Exp 3. Alizadeh A, Dyck SM, and Karimi-Abdolrezaee S. Traumatic Neurol 2012 Apr; 234(2): 362–372. spinal cord injury: an overview of pathophysiology, models 17. Kumar S, Jain S, Velpandian T, et al. Exposure to extremely and acute injury mechanisms. Front Neurol [Internet] low-frequency magnetic field restores spinal cord injury- 2019;10: 282. https://www.frontiersin.org/articles/10.3389/ induced tonic pain and its related neurotransmitter concentration fneur.2019.00282/full (cited June 2, 2020). in the brain. Electromagn Biol Med 2013 Dec; 32(4): 471–483. 4. Kumar S, Jain S, Behari J, et al. Effect of magnetic field on 18. Tong M, Qualls-Creekmore E, Browning KN, et al. Experimental food and water intake and body weight of spinal cord injured spinal cord injury in rats diminishes vagally-mediated gastric rats. Indian J Exp Biol 2010 Oct; 48(10): 982–986. responses to cholecystokinin-8s. Neurogastroenterol Motil Off 5. Das S, Kumar S, Jain S, et al. Exposure to ELF—magnetic J Eur Gastrointest Motil Soc 2011 Feb; 23(2): e69-79. field promotes restoration of sensori-motor functions in adult 19. Gondim FA, da-Graça JR, de-Oliveira GR, et al. Decreased rats with hemisection of thoracic spinal cord. Electromagn Biol gastric emptying and gastrointestinal and intestinal transits Med 2012 Sep; 31(3): 180–194. of liquid after complete spinal cord transection in awake rats. 6. Manjhi J, Kumar S, Behari J, et al. Effect of extremely low Braz J Med Biol Res Rev Bras Pesqui Medicas E Biol 1998 frequency magnetic field in prevention of spinal cord injury- Dec; 31(12): 1605–1610. induced osteoporosis. J Rehabil Res Dev 2013; 50(1): 17–30. 20. Giangregorio L and McCartney N. Bone loss and muscle 7. Ahmed Z, Wagdy M, Benjamin M, et al. Therapeutic effects of atrophy in spinal cord injury: epidemiology, fracture acrobatic exercise and magnetic field exposure on functional prediction, and rehabilitation strategies. J Spinal Cord Med recovery after spinal cord injury in mice. Bioelectromagnetics 2006; 29(5): 489–500. 2011 Jan; 32(1): 49–57. 21. Gater DR. Obesity after spinal cord injury. Phys Med Rehabil 8. Poirrier A-L, Nyssen Y, Scholtes F, et al. Repetitive transcranial Clin N Am 2007 May; 18(2): 333–351, vii. magnetic stimulation improves open field locomotor recovery 22. Fowler CJ, Griffiths D, and de Groat WC. The neural control of after low but not high thoracic spinal cord compression-injury micturition. Nat Rev Neurosci 2008 Jun; 9(6): 453–466. in adult rats. J Neurosci Res 2004 Jan 15; 75(2): 253–261. 23. de Groat WC and Yoshimura N. Mechanisms underlying the 9. Dey S, Bose S, Kumar S, et al. Extremely low frequency recovery of lower urinary tract function following spinal cord magnetic field protects injured spinal cord from the microglia- injury. Prog Brain Res 2006; 152: 59–84. and iron-induced tissue damage. Electromagn Biol Med 24. Morgan CW, de Groat WC, Felkins LA, et al. Axon collaterals 2017; 36(4): 330–340. indicate broad intraspinal role for sacral preganglionic neurons. 10. Basso DM, Beattie MS, and Bresnahan JC. Graded histological Proc Natl Acad Sci USA 1991 Aug 1; 88(15): 6888–6892. and locomotor outcomes after spinal cord contusion using the 25. Zehr EP and Duysens J. Regulation of arm and leg movement NYU weight-drop device versus transection. Exp Neurol 1996 during human locomotion. Neurosci Rev J Bringing Neurobiol Jun; 139(2): 244–256. Neurol Psychiatry 2004 Aug; 10(4): 347–361. 11. Wen J, Sun D, Tan J, et al. A consistent, quantifiable, and graded 26. 26. Barbour HR, Plant CD, Harvey AR, et al. Tissue sparing, rat lumbosacral spinal cord injury model. J Neurotrauma 2015 behavioral recovery, supraspinal axonal sparing/regeneration Jun 15; 32(12): 875–892. following sub-acute glial transplantation in a model of spinal 12. Wang C, Liu Y, Wang Y, et al. Low-frequency pulsed cord contusion. BMC Neurosci 2013 Sep; 14: 106. electromagnetic field promotes functional recovery, reduces 27. Hong JH, Bai DS, Jeong JY, et al. Injury of the spino-thalamo- inflammation and oxidative stress, and enhances HSP70 cortical pathway is necessary for central post-stroke pain. Eur expression following spinal cord injury. Mol Med Rep 2019 Neurol 2010; 64(3): 163–168. Mar; 19(3): 1687–1693. 56 Annals of Neurosciences 27(2) 28. Pal A, Singh A, Nag TC, et al. Iron oxide nanoparticles and 34. Kusuyama K, Tachibana T, Yamanaka H, et al. Upregulation magnetic field exposure promote functional recovery by of calcium channel alpha-2-delta-1 subunit in dorsal horn attenuating free radical-induced damage in rats with spinal cord contributes to spinal cord injury-induced tactile allodynia. transection. Int J Nanomedicine 2013; 8: 2259–2272. Spine J Off J North Am Spine Soc 18(6):1062–1069 29. Macias MY, Battocletti JH, Sutton CH, et al. Directed and 35. Grehl S, Viola HM, Fuller-Carter PI, et al. Cellular and enhanced neurite growth with pulsed magnetic field stimulation. molecular changes to cortical neurons following low intensity Bioelectromagnetics 2000 May; 21(4): 272–286. repetitive magnetic stimulation at different frequencies. Brain Stimulat 2015 Feb; 8(1): 114–123. 30. Mert T, Gunay I, Gocmen C, et al. Regenerative effects of pulsed magnetic field on injured peripheral nerves. Altern Ther 36. Sun Z, Ge J, Guo B, et al. Extremely low frequency Health Med 2006 Oct; 12(5): 42–49. electromagnetic fields facilitate vesicle endocytosis by increasing presynaptic calcium channel expression at a central 31. Ahmed Z and Wieraszko A. Combined effects of acrobatic synapse. Sci Rep 2016 Feb 18; 6: 21774. exercise and magnetic stimulation on the functional recovery after spinal cord lesions. J Neurotrauma 2008 37. Piacentini R, Ripoli C, Mezzogori D, et al. Extremely Oct; 25(10): 1257–1269. low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Ca(v)1-channel activity. J 32. Crowe MJ, Sun Z-P, Battocletti JH, et al. Exposure to pulsed Cell Physiol 2008 Apr; 215(1): 129–139. magnetic fields enhances motor recovery in cats after spinal cord injury. Spine 2003 Dec 15; 28(24): 2660–2666. 33. Manev H, Favaron M, Guidotti A, et al. Delayed increase of 2+ Ca influx elicited by glutamate: role in neuronal death. Mol Pharmacol 1989 Jul; 36(1): 106–112. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Neurosciences SAGE

Effect of Low Intensity Magnetic Field Stimulation on Calcium-Mediated Cytotoxicity After Mild Spinal Cord Contusion Injury in Rats

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© 2020 Indian Academy of Neurosciences (IAN)
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Abstract

Background: Magnetic field (MF) stimulation has the potential to reduce secondary damage and promote functional recovery after neural tissue injury. The study aimed to observe the effect of very low intensity (17.96µT) MF on general body condition, secondary damage, pain status, and locomotion. Methods: We exposed rats to MF (2 h/day × 3 weeks) after 6.25 mm contusion spinal injury. Locomotor behavior was evaluated by BBB score, pain assessment was done by recording threshold for tail flick, expression of voltage-gated calcium channels and extent of secondary damage in the spinal cord was assessed by immunofluorescence and Cresyl violet staining, respectively. Results: A significant (p ≤ .001) improvement in bladder function as well as BBB score was observed after MF exposure in comparison with sham and SCI over the observation period of 3 weeks. SCI group showed an increase in the threshold for vocalization after discharge, which decreased following MF exposure. Cresyl violet staining showed significantly higher tissue sparing (73%) at the epicenter after MF exposure when compared to SCI group. This was accompanied with a significant decrease in calcium channel expression in MF group as compared to SCI. Conclusion: The results suggest facilitation of sensory-motor recovery after MF exposure, which could be due to attenuation of secondary damage and calcium-mediated excitotoxicity in a mild contusion rat model of SCI. Keywords Spinal cord injury, contusion, magnetic field stimulation, locomotion, calcium, pain Abbreviations BBB: Basso, Beattie, and Bresnahan BMR: basal metabolic rate Ca: calcium ion CpG: central pattern generator DAPI: 4,6-diamidino-2-phenylindole FITC: fluorescein isothiocyanate K: potassium ion mA: milliampere MF: magnetic field Na: sodium ion PBS: phosphate buffer saline PBSTx: phosphate buffer saline triton SCI: spinal cord injury TTF: tail flick threshold VAD: vocalization after discharge VDCCs: voltage-dependent calcium channels µT: micro Tesla Faculty of Biological Sciences, University of Leeds, Woodhouse, Leeds, UK. Introduction Institute of Microbial Technology, Chandigarh, India Department of Physiology, All India Institute of Medical Sciences, New Delhi, India Spinal cord injury (SCI) initiates cascade of events, which Corresponding author: include significant electrolytic shifts, involving monovalent Suman Jain, Department of Physiology, All India Institute of Medical Sciences, + + 2+ (Na , K ) and divalent (Ca ) cations imbalance, that Ansari Nagar, New Delhi 110029, India. contributes to excitoxicity, oxidative stress, and degeneration E-mail: sumanjain10@gmail.com Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution- NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-Commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 50 Annals of Neurosciences 27(2) of neuronal tissue due to secondary injury progression. This MF Exposure Chamber leads to loss of sensory, motor, and autonomic functions in The MF exposure chamber (modified form of Helmholtz coil) body parts below the level of lesion. It also results in intense used has 18 and 8 turns of wire wound on circular formers chronic or neuropathic pain due to central sensitization. of two outer and inner electromagnetic coils, respectively. Ionic imbalance, specifically mitochondrial calcium overload Rats were kept in a polypropylene cage (50 × 20 × 15 cm3) immediately after SCI, seems to be one of the key players for on central area of the chamber that provides uniform MF of the neuronal death and glutamate-mediated neurotoxicity. 6,13 17.96µT, 50Hz. Transcranial as well as external magnetic field (EMF) stimulation has been shown to improve locomotor deficits, osteoporosis, tonic pain, general body conditions, lesion Procedure for Inducing Spinal Cord Injury 4–8 volume, density of serotonergic fibers at injury site, and Under anesthesia (thiosol; 50 mg/kg body weight, i.p.), attenuate inflammation and microglial activity in complete the dorsal surface of thoracic area was shaved and cleaned transection, hemisection, and compression rat models with povidone-iodine. An incision was made from the upper of SCI. A weight drop contusion rat model is one of the thoracic region to the upper lumbar region. Fascia and muscles most reproducible and reliable animal model for studying were removed from T11–T13 vertebral level (laminectomy) effectiveness of any therapeutic strategy in SCI as it closely to expose spinal T13 level of the cord (sham group). In SCI 10,11 mimics human injuries. Exposure of severely contused SCI and SCI + MF groups after laminectomy, mild contusion rats to pulsed EMF facilitates functional recovery by reducing injury (height of 6.25 mm) was done by New York University oxidative stress, inflammation, and increasing expression of (NYU) SCI contusion device. The body temperature was HSP70. However, the effect of MF on calcium-mediated maintained during surgery by controlled heating pads. cytotoxicity is not clear. Thus, objective of the study was to Suturing of muscle, fascia, and skin was done, and rats were observe the low intensity MF stimulation effect on general kept back in their home cages. To compensate for blood loss, body condition, locomotion, pain, and calcium imbalance in ringer lactate solution (5 mL, i.p.) was provided to rats along mild contusion SCI rats. with antibiotics (50 mg/kg b.wt Gentamycin, im; neosporin ointment applied locally) after surgery. We manually Methods expressed bladder 3 times/day until bladder control restored. The day of return of bladder function to normal was noted for Adult female Wistar rats weighing between 200 and 250 grams each rat of all the groups. were used for the study. Animals were obtained from Central Animal Facility (AIIMS, New Delhi, India) and kept in a room Assessment of Locomotor Functions having controlled temperature (25 ± 2 °C) and light: dark cycle (14:10 h). Ad libitum water and food pellets (Ashirwad The rat was placed in open field for 4 minutes to assess hind Industries Ropar, India) were provided. Committee for the limb movements using Basso, Beattie, and Bresnahan scoring Purpose of Control and Supervision of Experiments on Animals scale, which is based on movement of three hind limb joints (CPCSEA, Animal Welfare Division, Ministry of Environment, (hip, knee, and ankle). Video recording of the locomotor Forest and Climate Change, India) and Institutional Animal behavior of rat was done to perform off-line BBB scoring Ethics Committee (ethics no 966/IAEC/16) approved the between 0 and 21 scale by a blind observer. study. It was a 21 day protocol and after recording all the baseline behavioral recording, the animals were divided into Assessment of Sensory Functions three groups randomly: Sham (n = 6), only laminectomy was performed after anesthesia (Pentabaritol 50 mg/kg b.wt); Threshold for tail flick is a segmental response, which is SCI (n = 6): laminectomy was followed by contusion SCI; mediated via spinal cord. TTF was determined by applying AC SCI + MF (magnetic field exposure) (n = 6): one day after the current to tail of the rat. The rat was habituated in a Plexiglas contusion injury of spinal cord, the rats were exposed to MF restrainer for 30 minutes, before starting the experiment. Two (EMF, 50 Hz, 17.96µT, 2 h/d for 3 weeks). After 21 days of needle electrodes (0.5″ × 27G) were inserted intradermally, injury, all the rats were sacrificed (euthanatized) by transcardial approximately 2 mm deep and 2 cm apart into the middle perfusion under deep anesthesia. portion of the tail (rostrally and caudally) and were fastened Food intake, water intake, body weight, and BBB scoring with adhesive tape. In this test, noxious electrical stimulation were recorded on the 1st, 5th, 7th, 9th, 13th, and 21st day of was applied to stimulate nociceptive afferents (biphasic the study. The threshold for tail flick and vocalization after square wave pulse with frequency 40 Hz, 1.5 ms pulse discharge was quantified on 21st day. SCI + MF group of rats width and varying strength of current from 0.1 to 10 mA were kept in a polypropylene cages and placed in MF chamber (AD Instruments and Lab Chart 8 [MLS060]). The current for 2 h/day for 21 days. After transcardial perfusion, all the at which tail flick occurred was considered as the threshold spinal cord tissues were stored for histological analysis. for tail flick. Bhattacharyya et al. 51 16 rats (sham = 6, SCI = 5, SCI + MF = 4) survived the total Vocalization After Discharge study period of 21 days. The behavioral and histological data Electrical stimulus strength was increased beyond tail flick of these rats were used for analysis and are presented in the threshold in the same rat. A loud vocal sound of higher pitch results. that outlasted the duration of stimulus was noted as VAD. It There was no significant difference in baseline represents emotional component of pain, which is organized presurgery food intake of all the groups, whereas in limbic structure. following SCI food intake decreased significantly in all the groups (sham, p = .001; SCI, p = .001; SCI + MF, p = .033). Food intake was nadir immediately after surgery Histology and gradually increased over the days. In SCI group this After three weeks of surgery, the rats were deeply decrease was maintained till day 5 (p = .039), whereas in anesthetized by injecting thiosol (i.p., 50 mg/kg of body SCI + MF group, no statistically significant difference in weight) and perfused transcardially with cold saline (4 °C) food intake on any of the postoperative days was observed and 4% paraformaldehyde. The spinal cord along with when compared with basal. Intergroup analysis revealed the vertebral column was isolated from T5 to L5 and post a significant decrease in food intake in SCI (p = .02) and fixed with 4% paraformaldehyde at 4 °C. A 6 mm long SCI + MF (p = .03) groups as compared to sham (Figure spinal cord tissue containing lesion epicenter was taken, 1) on day 21 and significant decrease in SCI (p = .001) cryosectioned (16µm thick), and mounted on poly-L-lysine as compared to SCI + MF on day 5. Body weight did not coated slides. Cresyl violet staining of the sections was done change significantly among all the groups after surgery using standard protocol to assess the lesion area extent and (Figure 2). volume. The total area of injury in one section was tracked by light microscope and calculated using NIS-Element Figure 1. Food Intake (gm, Mean ± SD) of All the Rats of All Groups software (Nikon). Immunohistochemistry for L-Type Calcium Channels Sections were collected on poly-L-lysine coated slides and washed with PBSTx three times each for 5 minutes. After that for 2 hours, 10% normal goat serum was added on to the sections for blocking. Then, they were incubated in primary antibody (anti-L-type calcium channel antibody C1353 at concentration 1:200) overnight at 4°C. It was again washed with PBSTx five times each for 3 minutes and then incubated with secondary antibody (fluorescein isothiocyanate conjugated at concentration of 1:400) for 2 hours at room Surce: Authors own. temperature. Finally washing with PBSTx five times for 3 Note: # indicates sham vs SCI/SCI + MF; Food intake increased significantly minutes was done. Slides were mounted in 4,6-diamidino-2- after MF stimulation. phenylindole (DAPI) containing mounting media. Figure 2. Body Weight (Mean ± SD) of All Groups at Different Time Points Statistical Analysis Statistical analysis was performed using Statistical Package for Social Sciences version 15 software (SPSS Inc, Chicago, IL, USA). The data are presented as the mean ± standard deviation. Data of each time point between the groups was compared by the Kruskal–Wallis test with post hoc analysis done by using Dunn’s test with the Bonferroni correction. Statistical significance was accepted at p ≤ .05. Results General Body Condition A total of 18 rats were taken and after surgery, randomly Source: Authors own. divided into three groups (n = 6 rats in each group). A total of Note: No significant change was observed throughout the study period . 52 Annals of Neurosciences 27(2) Figure 3. Day of Restoration of Urinary Bladder Function (Mean Figure 4. Effect of MF Exposure on BBB Score (Mean ± SD) in All ± SD) the Rats at All the Time Points Source: Authors own. Note: # indicates sham vs SCI/ SCI + MF; * indicates SCI vs SCI + MF. A significant improvement in BBB score was observed following MF exposure. Figure 5. Effect of MF Exposure on Threshold for Tail Flick and Vocalization After Discharge (mA, Mean ± SD) Source: Authors own. Note: # indicates sham vs SCI/SCI + MF; * indicates SCI vs SCI + MF. Spontaneous evacuation of bladder was achieved significantly earlier in SCI + MF group. After SCI, there was loss of urinary bladder function in both SCI and SCI + MF groups. Manual evacuation was done eight hourly to survive the rats. A significant effect of intervention was observed on the day of restoration of Source: Authors own. spontaneous bladder function (χ = 12.54; p = .001). In SCI Note: No significant change observed in phasic pain after mild SCI and MF group, post hoc analysis showed that the day of spontaneous exposure. evacuation of bladder was achieved significantly later (day 6.2 ± 1.48, p = .0001) in comparison to sham (day 1.6 ± 0.5). MF exposure had significant effect and in SCI + MF group, Pain Threshold bladder function recovered significantly (day 3.25 ± 0.5; The sensory as well as emotional components of pain were p = .0002) earlier in comparison to SCI group (Figure 3). assessed by recording threshold for tail flick and vocalization after discharge. There was no significant (χ = 4.05, p = Locomotor BBB Score .13) difference in the threshold for tail flick among all the three groups (Figure 5). However, on post hoc analysis, on Bilateral hind limb paralysis occurred in all the rats of all the day 21, a significant increase in SCI + MF group (p = .03) groups after spinal cord contusion injury at thoracic level. was observed as compared to sham. When vocalization after BBB score was significantly different (p ≤ .001) among all discharge threshold was compared between groups, there was the groups following surgery. In SCI + MF and SCI groups, a trend for an increase of the threshold in SCI group and a a significant drop of BBB score was observed after injury decrease in MF exposure group, but it was not statistically from the basal as well as in comparison to sham group. significant ( χ = 2.65, p = .26). Over the observation period of 3 weeks, the score increased gradually in both the groups but was maintained at a lower level (p ≤ .001) in comparison with sham. MF stimulation Histology and Immunohistochemistry significantly improved hind limb function starting from day Morphological analysis of the spinal cord revealed presence 1 of surgery. In MF group, the BBB score was significantly of lesion in the center and sparing at the periphery of the tissue. higher (p ≤ .001) at all time points during 3-week study In Cresyl violet staining, the central lesion area depicted period as compared to SCI group, suggesting beneficial effect cyst formations, gliosis, zones of partial demyelination, of intervention (Figure 4). Bhattacharyya et al. 53 microcysts, scarring, and was mostly localized to dorsal horn. Discussion In SCI group, 51% fibers were spared, whereas following intervention of MF exposure in SCI + MF group, it increased SCI leads to intolerable chronic pain besides severe deficits in 15,16 to 73%. A statistically significant (χ = 8.43, p = .004) motor, autonomic, and sensory functions. Chronic pain is difference in total damage area of spinal cord was observed an unavoidable part of secondary cascade, which ensues after when comparison was done between SCI + MF and spinal cord injury subsidies and is resistant to analgesics. This SCI groups. A significant decrease in the damaged area gives a reason to find an intervention that could effectively following MF exposure for 3 weeks was evident (Figure treat chronic pain. In the recent years, magnetic field has 6). Immunohistochemistry for L-type voltage-dependent shown to improve sensorimotor function, tonic pain and calcium channels (VDCCs) in spinal cord tissue sections was hyperalgesia in complete transection/hemisection models of 4,5,17 performed to assess role of calcium in excitotoxicity. DAPI SCI. The current study was designed to understand low staining revealed calcium localization adjacent to the nucleus, intensity electromagnetic stimulation effect on locomotion, on the cell membrane. A significantly higher expression of chronic pain, and on calcium signaling in mild contusion rat L-type VDCCs in SCI group was evident in comparison to model of SCI. We used a mild thoracic contusion injury model sham. However, we observed that in the SCI + MF group, the in our study as it mimics best the human condition among expression decreased in comparison to SCI (Figure 7). all the other SCI models available. SCI is generally followed by a cascade of events including generation of free radical, Figure 6. (A) Representative Images of Cresyl Violet Staining of ionic imbalance and alteration of cellular events which lead to Spinal Cord in Each Group. (B) Effect of MF exposure on Mean degeneration of cord both rostrally and caudally and thereby (±SD) Damaged and Spared Area in All the Rats. disruption in sensory and motor function. We also observed bilateral hind limb paralysis and loss of bladder function (A) immediately after contusion at T13 in all the rats. Food intake was significantly decreased in spinal cord (B) injured rats. Consistent decrease in food intake was observed till fifth day of injury in SCI group, following which it recovered spontaneously. Since SCI is a type of neurotrauma, thereby a stressful event, initial decrease in feeding behavior in our study was expected. The decrease in body weight and feeding behavior may also be secondary to alterations in gastric 18,19 motility and emptying. During the initial weeks after SCI, even the patients have been shown to have a tendency to lose weight due to muscle atrophy and bone mineral loss resulting in fat free mass loss and lower body mass index. Reduced fat-free mass, sympathetic blunting, cardiopulmonary Note: * indicates SCI vs SCI + MF. A significant decrease in damaged tissue area and an increase in spared tissue area is observed after MF exposure. dysfunction, work capacity reductions, and diminished anabolic hormones also sustain the BMR decrement after the Figure 7. Effect of magnetic field stimulation on voltage-gated cal- acute phase of SCI. In SCI rats, urinary bladder function cium channel expression in spinal cord tissue. The expression was remained disrupted for significantly higher time as compared decreased in MF group as compared to SCI. to sham in the present study. Animal studies have exhibited that reflex micturition is mediated by a spinobulbospinal pathway, whereas urethral relaxation is brought about by excitation of the pontine micturition center, which in turn activates descending pathways. This relaxation eventually is followed by an activation of sacral parasympathetic outflow (L6–S1 in rats), which leads to contraction of the bladder, an increase in intravesical pressure and the flow of urine. Thus, SCI at the thoracic level eliminates voluntary and supraspinal control of voiding, leading to an initial phase of areflexic bladder and complete urinary retention. Finally, there is a slow development of automatic micturition and 23,24 bladder hyperactivity mediated by spinal reflex pathways. Thoracic SCI also can cause complete paralysis or motor deficits in the hind limbs depending on the severity of the injury. Paraplegia was observed immediately following mild thoracic injury in the present study, which gradually partially 54 Annals of Neurosciences 27(2) recovered over the observation period of 21 days postinjury, Voltage-gated calcium channels mediate calcium influx into though it was significantly less than sham group, as assessed living cells upon membrane depolarization and have a critical by BBB score. This gradual recovery in locomotor function is role in calcium signaling. Increase in alpha-2-delta subunit possibly because of the sparing of central pattern generators, of voltage sensitive calcium channel in SCI rats has been which is located at L1–L2 segment in rat, and we gave shown to correlate with development of tactile allodynia of lesion at T13 spinal segment. Further, 51% tissue was spared hind paw. MF stimulation (17.96µT, 50 Hz, 2hrs/day for 3 in the SCI rats, sprouting from them may have contributed weeks) of spinal cord of injured rats, in the present study, to the spontaneous recovery of BBB score. Spared area after reduced the calcium channel expression. In literature, there SCI is a prominent marker for tissue integrity and behavioral are contradictory reports on the effects of EMF on calcium recovery. channels. Low frequency-EMF, 1–25 Hz for <180 ms, with Chronic pain is among the most prominent characteristic 1–2 T intensity reduced the calcium transit from endoplasmic feature of SCI; however, mild injury to the cord could not reticulum in F-11 cells. Sun et al. (2016) reported enhanced alter pain parameters significantly in our study. Spinothalamic calcium channel expression of all the subtypes at presynaptic pathway, which is mainly located in the ventral spinal cord, terminal in situ brain slice preparation, thereby enhancing is majorly responsible for the pain perception. In mild SCI, vesicle endocytosis and synaptic plasticity following there is major damage to dorsal part, mainly consisting of exposure to low frequency MF (uniform 1 mT MF with 50 Hz gracile fasciculus and cuneate fasciculus. These are the first- frequency) for 8–10 days. Low frequency electromagnetic order sensory neurons that relay proprioceptive, fine touch, field (1 mT, 50 Hz) stimulation also enhanced neurogenesis and vibration sensations to the thalamus, thereby the pain via expression of calcium receptor (Ca 1). It is therefore sensation remains unaffected. Under Cresyl violet staining suggested that the expression of calcium channels following of the lesioned spinal cord tissue, we also observed major MF stimulation may depend upon the model used (in vivo or damage to the both fasciculi and sparing of dorsal horn as in vitro), magnetic stimulation parameters, and the design of well as ventral and lateral parts of the spinal cord. the study protocol. MF exposure for 5–8 weeks has been shown to ameliorate locomotor deficits, osteoporosis, tonic pain, and general body Conclusion conditions in completely transected or hemisected spinal cord 5,6,9,17 rat models. MF exposure, for 3 weeks to rats with mild The present study shows beneficial role of short-term MF spinal contusion injury, was able to improve general body exposure in the contusion SCI rat model on locomotor condition like food intake and urinary bladder function. This behavior by preventing calcium-mediated excitotoxicity. is in agreement with previous literature, wherein MF exposure has been shown to improve general body condition and Acknowledgments facilitate early recovery of urinary bladder function. There We would like to thank Mr Pursottam Samal for histology and was also a significant improvement in BBB locomotor scale Department of Biotechnology, AIIMS, for all the support during as compared to SCI, though it was lower than sham group. this research work. We acknowledge the support rendered by Dr This may be due to 73% sparing of neural tissue, which under Anushree Gupta, Associate Professor, AIIMS, Delhi. the influence of magnetic fields was able to regenerate and compensate for the lost tissue. MF exposure has been shown Author Contributions to activate regenerative processes under both in vitro and in 29,30 vivo conditions. It also has proved beneficial in restoring S. J. Conceived the project and majority of the laboratory work has been performed in her laboratory. locomotion, muscle contraction properties, and limiting S. B. and S. S. Performed all the experimental works. muscle degeneration in SCI rats. In total, 5%–10% fibers at S. B. Wrote the manuscript also. the epicenter of contusion (T7–9) and clip compression (T1) S. K. Standardized and helped in performing behavioral experiments. injuries of spinal cord and 1%–5% descending axons have been shown to be sufficient to restore the segmental local Declaration of Conflicting Interests 31,32 circuits necessary for locomotion. After SCI due to ionic imbalance and excitotoxicity of The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. glutamate receptor, calcium concentrations reach critical level, which leads to neuronal loss. We studied L-type Ethical Statement VDCCs, which is a marker of excitotoxicity in neuronal cells to unravel the underlying cellular mechanism of the The study was approved by the Committee for the Purpose of Control beneficial effects of MF. A significantly higher expression was and Supervision on Experiments on Animals (CPCSEA), New observed in SCI as compared to sham and SCI + MF groups. Delhi, Animal Welfare Division under Ministry of Environment, High levels of glutamate cause excitotoxicity by facilitating Forest and Climate Change (Govt. of India) and approved by the 2+ 33 Institutional Animal Ethics Committee (Ethics no 966/IAEC /16). entry of high levels of calcium ions (Ca ) to enter the cell. Bhattacharyya et al. 55 Funding 13. Kirschvink JL. Uniform magnetic fields and double- wrapped coil systems: improved techniques for the design The authors received no financial support for the research, of bioelectromagnetic experiments. Bioelectromagnetics authorship, and/or publication of this article. 1992; 13(5): 401–411. 14. Mukherjee K, Mathur R, and Nayar U. Ventromedial Reference hypothalamic mediation of sucrose feeding induced pain 1. Hulsebosch CE. Recent advances in pathophysiology and modulation. Pharmacol Biochem Behav 2001 Jan; 68(1): 43–48. treatment of spinal cord injury. Adv Physiol Educ 2002 15. Morgan D, Carter CS, DuPree JP, et al. Evaluation of Dec; 26(1–4): 238–255. prescription opioids using operant-based pain measures in rats. 2. Lindsey AE, LoVerso RL, Tovar CA, et al. An analysis of Exp Clin Psychopharmacol 2008 Oct; 16(5): 367–375. changes in sensory thresholds to mild tactile and cold stimuli 16. Gwak YS, Kang J, Unabia GC, et al. Spatial and temporal after experimental spinal cord injury in the rat. Neurorehabil activation of spinal glial cells: role of gliopathy in central Neural Repair 2000; 14(4): 287–300. neuropathic pain following spinal cord injury in rats. Exp 3. Alizadeh A, Dyck SM, and Karimi-Abdolrezaee S. Traumatic Neurol 2012 Apr; 234(2): 362–372. spinal cord injury: an overview of pathophysiology, models 17. Kumar S, Jain S, Velpandian T, et al. Exposure to extremely and acute injury mechanisms. Front Neurol [Internet] low-frequency magnetic field restores spinal cord injury- 2019;10: 282. https://www.frontiersin.org/articles/10.3389/ induced tonic pain and its related neurotransmitter concentration fneur.2019.00282/full (cited June 2, 2020). in the brain. Electromagn Biol Med 2013 Dec; 32(4): 471–483. 4. Kumar S, Jain S, Behari J, et al. Effect of magnetic field on 18. Tong M, Qualls-Creekmore E, Browning KN, et al. Experimental food and water intake and body weight of spinal cord injured spinal cord injury in rats diminishes vagally-mediated gastric rats. Indian J Exp Biol 2010 Oct; 48(10): 982–986. responses to cholecystokinin-8s. Neurogastroenterol Motil Off 5. Das S, Kumar S, Jain S, et al. Exposure to ELF—magnetic J Eur Gastrointest Motil Soc 2011 Feb; 23(2): e69-79. field promotes restoration of sensori-motor functions in adult 19. Gondim FA, da-Graça JR, de-Oliveira GR, et al. Decreased rats with hemisection of thoracic spinal cord. Electromagn Biol gastric emptying and gastrointestinal and intestinal transits Med 2012 Sep; 31(3): 180–194. of liquid after complete spinal cord transection in awake rats. 6. Manjhi J, Kumar S, Behari J, et al. Effect of extremely low Braz J Med Biol Res Rev Bras Pesqui Medicas E Biol 1998 frequency magnetic field in prevention of spinal cord injury- Dec; 31(12): 1605–1610. induced osteoporosis. J Rehabil Res Dev 2013; 50(1): 17–30. 20. Giangregorio L and McCartney N. Bone loss and muscle 7. Ahmed Z, Wagdy M, Benjamin M, et al. Therapeutic effects of atrophy in spinal cord injury: epidemiology, fracture acrobatic exercise and magnetic field exposure on functional prediction, and rehabilitation strategies. J Spinal Cord Med recovery after spinal cord injury in mice. Bioelectromagnetics 2006; 29(5): 489–500. 2011 Jan; 32(1): 49–57. 21. Gater DR. Obesity after spinal cord injury. Phys Med Rehabil 8. Poirrier A-L, Nyssen Y, Scholtes F, et al. Repetitive transcranial Clin N Am 2007 May; 18(2): 333–351, vii. magnetic stimulation improves open field locomotor recovery 22. Fowler CJ, Griffiths D, and de Groat WC. The neural control of after low but not high thoracic spinal cord compression-injury micturition. Nat Rev Neurosci 2008 Jun; 9(6): 453–466. in adult rats. J Neurosci Res 2004 Jan 15; 75(2): 253–261. 23. de Groat WC and Yoshimura N. Mechanisms underlying the 9. Dey S, Bose S, Kumar S, et al. Extremely low frequency recovery of lower urinary tract function following spinal cord magnetic field protects injured spinal cord from the microglia- injury. Prog Brain Res 2006; 152: 59–84. and iron-induced tissue damage. Electromagn Biol Med 24. Morgan CW, de Groat WC, Felkins LA, et al. Axon collaterals 2017; 36(4): 330–340. indicate broad intraspinal role for sacral preganglionic neurons. 10. Basso DM, Beattie MS, and Bresnahan JC. Graded histological Proc Natl Acad Sci USA 1991 Aug 1; 88(15): 6888–6892. and locomotor outcomes after spinal cord contusion using the 25. Zehr EP and Duysens J. Regulation of arm and leg movement NYU weight-drop device versus transection. Exp Neurol 1996 during human locomotion. Neurosci Rev J Bringing Neurobiol Jun; 139(2): 244–256. Neurol Psychiatry 2004 Aug; 10(4): 347–361. 11. Wen J, Sun D, Tan J, et al. A consistent, quantifiable, and graded 26. 26. Barbour HR, Plant CD, Harvey AR, et al. Tissue sparing, rat lumbosacral spinal cord injury model. J Neurotrauma 2015 behavioral recovery, supraspinal axonal sparing/regeneration Jun 15; 32(12): 875–892. following sub-acute glial transplantation in a model of spinal 12. Wang C, Liu Y, Wang Y, et al. Low-frequency pulsed cord contusion. BMC Neurosci 2013 Sep; 14: 106. electromagnetic field promotes functional recovery, reduces 27. Hong JH, Bai DS, Jeong JY, et al. Injury of the spino-thalamo- inflammation and oxidative stress, and enhances HSP70 cortical pathway is necessary for central post-stroke pain. Eur expression following spinal cord injury. Mol Med Rep 2019 Neurol 2010; 64(3): 163–168. Mar; 19(3): 1687–1693. 56 Annals of Neurosciences 27(2) 28. Pal A, Singh A, Nag TC, et al. Iron oxide nanoparticles and 34. Kusuyama K, Tachibana T, Yamanaka H, et al. Upregulation magnetic field exposure promote functional recovery by of calcium channel alpha-2-delta-1 subunit in dorsal horn attenuating free radical-induced damage in rats with spinal cord contributes to spinal cord injury-induced tactile allodynia. transection. Int J Nanomedicine 2013; 8: 2259–2272. Spine J Off J North Am Spine Soc 18(6):1062–1069 29. Macias MY, Battocletti JH, Sutton CH, et al. Directed and 35. Grehl S, Viola HM, Fuller-Carter PI, et al. Cellular and enhanced neurite growth with pulsed magnetic field stimulation. molecular changes to cortical neurons following low intensity Bioelectromagnetics 2000 May; 21(4): 272–286. repetitive magnetic stimulation at different frequencies. Brain Stimulat 2015 Feb; 8(1): 114–123. 30. Mert T, Gunay I, Gocmen C, et al. Regenerative effects of pulsed magnetic field on injured peripheral nerves. Altern Ther 36. Sun Z, Ge J, Guo B, et al. Extremely low frequency Health Med 2006 Oct; 12(5): 42–49. electromagnetic fields facilitate vesicle endocytosis by increasing presynaptic calcium channel expression at a central 31. Ahmed Z and Wieraszko A. Combined effects of acrobatic synapse. Sci Rep 2016 Feb 18; 6: 21774. exercise and magnetic stimulation on the functional recovery after spinal cord lesions. J Neurotrauma 2008 37. Piacentini R, Ripoli C, Mezzogori D, et al. Extremely Oct; 25(10): 1257–1269. low-frequency electromagnetic fields promote in vitro neurogenesis via upregulation of Ca(v)1-channel activity. J 32. Crowe MJ, Sun Z-P, Battocletti JH, et al. Exposure to pulsed Cell Physiol 2008 Apr; 215(1): 129–139. magnetic fields enhances motor recovery in cats after spinal cord injury. Spine 2003 Dec 15; 28(24): 2660–2666. 33. Manev H, Favaron M, Guidotti A, et al. Delayed increase of 2+ Ca influx elicited by glutamate: role in neuronal death. Mol Pharmacol 1989 Jul; 36(1): 106–112.

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Annals of NeurosciencesSAGE

Published: Apr 1, 2020

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