Daily Body Clocks May Influence How Pulsed Electromagnetic Fields Help Spinal Disc Damage in Rats

PEMF stimulators were obtained from GHY-III, Air Force Military Medical University (AMMU), Xi'an, China; China Patent no. ZL02224739.4 [32, 33]. To conduct animal experiments, we used a PEMF generator and three Helmholtz coils of 800 mm diameters. Specifically, the coils were located coaxially with an interval of 304 mm, and each coil consisted of enameled coated copper wire, with a diameter of 0.8 mm. The PEMF waveform comprised of a pulsed burst (burst width 5 ms; pulse width, 0.2 ms; pulse wait, 0.02 ms; burst wait, 60 ms; pulse rise and fall times: 0.3 μs, 2.0 μs) repeated at 15 Hz. This particular waveform was shown to promote nerve and bone regeneration in previous studies and was similar to the setting of PEMF in treating IVDD [30, 31, 34]. An oscilloscope (6000 series; Agilent Technologies, USA) was used to visualize wave shape and frequency and a Gaussmeter (Model 455 DSP; Lake Shore Cryotronics) for monitoring the accuracy of PEMF output. The rats in the treatment groups received PEMF stimulation 4 hours per day immediately after surgery. In case of cellular experiments, different Helmholtz coils were used, which was composed of two coils at a 100 mm interval distance.

All animal experiments followed the animal care guidelines approved by the Animal Research Committee of the Fourth Military Medical University. Twenty, 3-month-old male Sprague-Dawley rats were selected (weight: 260-280 g, from Laboratory Animal Center of FMMU). Water and food were given ad libitum. The temperature was maintained at 24 ± 2°C, with 50–60% humidity. The light schedule included 12 hours of daylight from 7: 00 AM and 12 hours of darkness from 7:00 PM. The rats were conditioned to the laboratory and observation conditions ≥ 30 min a day for 7 days prior to experimentation. All rats were anesthetized intraperitoneally with pentobarbital solution (1%, 40 mg/kg). Rats were arbitrarily separated into 4 groups: sham (n = 5); needle puncture (n = 5); needle puncture with daytime (7:00-11:00) PEMF treatment, DPEMF (n = 5); and needle puncture with nighttime (19:00-2300) PEMF treatment, NPEMF (n = 5). As previous studies described [35, 36], after disinfecting the tail skin with iodophor and alcohol, a 20-gauge needle was utilized to fully puncture tail disc from Co6 to Co10 levels; the needle was rotated 180 and left for 60 seconds before removal. After the operation, iodophor was used to disinfect the stab site. In the sham group (n = 5), the rats were placed under anesthesia without needle puncture injuries. Following the operation, all the animals were brought back to their cages and given food and water ad libitum. The rats in the experimental group were exposed to PEMF treatment (4 h/d) immediately after surgery. According to previous studies, after 8 weeks of treatment, rats were sacrificed and tested immediately [35, 36]. Before specimen harvest, rats were sent to take X-ray and MRI for the disc height and IVDD grade analysis. The Co6/Co7 discs, including endplate and bone, were excised and fixed in 4% paraformaldehyde for histological staining. The Co7/Co8 and Co8/Co9 discs were collected and instantly frozen in liquid nitrogen before storage at -80°C for transcriptome sequencing (mRNA-seq) and real-time RT-PCR. The Co9/Co10 disc was excised and maintained at -80°C for subsequent analysis.

10 male Sprague-Dawley rats (3-4 weeks old) were euthanized with an overdose of 1% sodium pentobarbital solution and NP cells were isolated, according to prior reports [37, 38]. In brief, the rat tail was removed under aseptic conditions, NP tissues were collected under a stereo microscope, digested in 0.1% type II collagenase for 4 hours at 37°C, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the pellet was resuspended in DMEM/F12 (1 : 1) medium (with 10% FBS, 1% P/S). Finally, the suspension was transferred to a culture flask and cultured in an incubator containing 5% CO₂ at 37°C. Cells from passage 2 to 4 were used for subsequent experimentation. In the first set of the in vitro studies, NP cells were exposed to PEMF for 4 hours, and cellular toxicity was assessed via flow cytometry at four separate times (0 h, 24 h, 48 h, and 72 h). In the second part of in vitro studies, cells received serum shock to induce expression of clock genes, as previously reported [39–41]. In brief, cells were starved for 48 h before stimulation with serum-rich medium (50% DMEM/F12 and 50% fetal bovine serum) for an additional 2 h. Subsequently, following DMEM/F12 (without serum) wash, cells were grown in serum-free DMEM/F12. NP cells were then harvested after 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, and 48 h from the initiation of serum shock. To induce PEMF stimulation, NP cells were treated with PEMF at time points 8 h and 20 h, respectively, for 4 h. After 24 h incubation, the cells were collected for molecular expression evaluation.

Rat X-ray and MRI images were obtained 8 weeks posttreatment. X-ray imaging of rat tail discs was performed using an X-ray system (Faxitron Bioptics, LLC, Wheeling, IL, USA). The disc height index (DHI) was computed by two blinded spinal surgeons, as described previously [42]. Alterations in DHI was represented by % DHI, which were standardized with preoperative DHI (%DHI = 100 × postoperative DHI/preoperative DHI). MRI images were performed on an MRI system (Siemens 3T Magnetom Trio Tim scanner, Munich, Germany). The IVDD score was also calculated by two spinal surgeons, based on the Pfirrmann grading system, ranging from grade I to grade IV [43].

Following 8 weeks of treatment, rats were sacrificed, and subsequently, the tail vertebrae (Co6-Co7) were harvested and fixed in 4% paraformaldehyde. Prior to histological processing, samples were scanned with micro-CT (eXplore Locus SP; GE, Fairfield, US) using X-ray tube settings of 80 kV and 80 mA and exposure time of 3000 ms. The scan resolution was adjusted to 14 mm, voxel size to 27 mm, and, thresholding value to 1150. The reconstructed images were generated via the Microview v1.1.1 software (GE Medical Systems). The bone volume/tissue volume (BV/TV) of the upper and lower endplates of each IVD was calculated.

Upon fixation in 4% paraformaldehyde, the IVD tissue was decalcified, paraffin-embedded, and sliced into 7 μm thick sagittal sections before staining with Hematoxylin & Eosin (H&E), Safranin O-Fast Green (SOFG), and Sirius red. The histological score was assessed by blinded independent researchers, as described previously [43, 44].

IVD morphology was assessed with a SEM (SEM; JSM-4800; Hitachi). In short, an IVD was cut along the coronal plane, fixed in 2.5% glutaraldehyde solution for 24 hours, and dried under partial vacuum. Then, the disc was sputter coated with gold and analyzed under accelerating voltage of 5-10 kV.

Total disc RNA was isolated and analyzed using Sinotech Genomics (Shanghai, China). Next, mRNA differential expression analysis was conducted with the R package edgeR. Differentially expressed RNAs with ∣log₂(FC) | value > 1 and q value < 0.05 were deemed significantly regulated and underwent additional analysis. Gene Ontology (GO) analysis for biological processes, cellular components, and molecular function as well as KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomes http://www.genome.ad.jp/kegg↗) was also performed via the enrich R package.

Total RNA was isolated from disc tissue samples (both NP and AF tissue) via the Total RNA Mini Kit (QIAGEN, Germany), following kit directions, and then converted to cDNA, prior to qPCR analysis via SYBR Green PCR Master Mix (TAKARA, Japan) and a Bio-Rad CFX96 PCR System (Bio-Rad, Australia). GAPDH was used as an internal reference. The primers were listed as follows: collagen II (F) 5′-CGAGGCAGACAGTACCTTGA-3′, (R) 5′-TGCTCTCGATCTGGTTGTTC-3′; Aggrecan (F) 5′-CTTCCCAACTATCCAGCCAT-3′, (R) 5′-TCACACCGATAGATCCCAGA-3′; MMP3 (F) 5′-GCTCATCCTACCCATTGCAT-3′, (R) 5′-GCTTCCCTGTCATCTTCAGC-3′; GAPDH (F) 5′-GGCACAGTCAAGGCTFAGAATG-3′, (R) 5′-GGTGGTGAAGACGCCAGTA-3′; Bmal1 (F) 5′-TCCGATGACGAACTGAAACAC-3′, (R) 5′-CTCGGTCACATCCTACGACAA-3′; Per2 (F) 5′-AGTGACGGGTCGAGCAAAG-3′, (R) 5′-TCATGTCGGGCTCTGGAATG-3′; Clock (F) 5′-GAACTTGGCGTTGAGGAGTCT-3′, (R) 5′-GTGATCGAACCTTTCCAGTGC-3′; Cry1 (F) 5′-GTCCAGCGACAGAGCAGTAAC-3′; (R) 5′-TACTGACTCTCCCACCAACTTCA-3′; Rev-verα (F) 5′-GGAGGTGGTAGAATTTGCCAA-3′, (R) 5′-CCTTCACGTTGAACAATGACG-3′.

The IVD tissue was homogenized with a tissue homogenizer (Servicebio, China), before lysis with RIPA buffer (Beyotime) containing phosphatase and protease inhibitors for 10 minutes. The NP cells were directly lysed with RIPA lysate, and the resulting lysate underwent centrifugation at 12,000 g for 15 minutes. Protein quantification was done with a BCA analysis kit. The samples were next boiled for 15 minutes and the proteins were separated via SDS-PAGE, before transfer to PVDF membranes (Thermo Fisher Scientific). The membranes underwent blocking in 5% nonfat milk in TBS-Tween (TBST) at room temperature (RT) for 1 h, before overnight (ON) incubation with primary antibodies at 4°C. The primary antibodies used were anti-collagen II (1 : 1000, abcam) and MMP-3 (1 : 1000, abcam). After ON incubation, the membranes were rinsed three times in TBST and incubated with secondary HRP-linked antibodies for 1 h. Finally, the immunoreactive bands were assessed via chemiluminescent analysis (Bio-Rad).

To measure SOD and MDA, two common indicators of oxidative stress, IVD tissue was homogenized and centrifuged at 900 g at 4°C for 15 min, and supernatant was extracted. According to the manufacturer's instructions (Beyotime Biotechnology Co., LTD., Shanghai, China), the supernatant and standard substance were added into the colorimetric dish, and the OD value (530 nm) was recorded. SOD and MDA levels were calculated according to the standard curve.

NP cell fixation was done in 4% paraformaldehyde for 10-15 minutes before permeabilizing with 0.2% Triton X-100 for 20 minutes. The cells underwent blocking in 10% normal goat serum at RT for 1 h, before incubation in primary antibodies against anti-collagen II (1 : 200) and MMP-3 (1 : 150) at 4°C. Next, cells were placed in secondary antibodies (FITC or Cy-3, 1 : 200) for 1 h at RT and finally incubated with DAPI for 10 min. Each step was followed by an additional 3 washing steps (15 min) with PBS. Cells were visualized under a fluorescence microscope, and fluorescence intensity quantification was done with ImageJ software.

Data analyses were done with the SPSS 22.0 software (SPSS Inc., Chicago, IL). One way ANOVA or Student's t-test was used to identify significant differences (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001). Data were expressed as mean with error bars denoting standard deviation.

The rat tail needle puncture model representing IVDD was used to evaluate the therapeutic effect of CR and PEMF cotherapies on IVDD. As shown in Figure 1(a), loss of IVD height, scanned by X-ray, was observed in all puncture and treatment groups. In the DPEMF group, the disc height was markedly elevated than in the NPEMF rats (P < 0.05, Figures 2(a) and 2(b)), both of which were superior to the puncture rats (Figures 2(a) and 2(b)). In addition, the bone volumes (BV/TV) of the upper and lower endplate vertebral bodies were analyzed by micro-CT 3D reconstruction. Based on our results, both PEMF groups exhibited significantly lower BV/TV, compared to the puncture rats (P < 0.01, DPEMF vs. puncture; P < 0.05, NPEMF vs. puncture). Although there was a decreasing trend in the DPEMF group (Figures 2(c) and 2(d)), no obvious difference was seen between the DPEMF and NPEMF groups. Further MRI studies (Figure 2(e)) revealed that the IVD Pfirrmann grading score in the DPEMF rats was much lower, compared to the NPEMF (P < 0.01, Figures 2(e) and 2(f)) and puncture rats (P < 0.01 Figures 2(e) and 2(f)).

Histological examinations were performed to evaluate the morphological changes after PEMF treatment. H&E (Figure 3(a)) and SOFG (Figure 3(b)) staining showed normal morphology and structure in the sham group, including proteoglycan-rich NP tissue, well-arranged AF, complete cartilage endplates, and clear AF-NP boundary. In the puncture group, a small number of irregularly shaped NP cells were seen, with fiber lamellae protruded inwardly in the AF, while the endplate was partially disrupted, and the AF-NP boundary was unclear. In the DPEMF treatment group, parts of the NP tissue were restored, and AF lamellae protruded inward in a moderately compensatory manner. Conversely, in the NPEMF treatment group, a small part of the NP tissue was left, and the AF structure appeared less disordered than in the puncture group. The histological score was highest in the puncture group, followed by NPEMF group, and then the DPEMF and sham groups (Figure 3(d)), indicating that the DPEMF was superior to NPEMF in maintaining IVDD histology.

Sirius red staining (Figure 3(c)) under polarized light showed that type I and type III collagens were orderly arranged and NP (mainly contain type II collagens) in the center were totally black in the IVDs of the sham group. During IVDD, however, the type I and type III collagens gradually replaced type II collagen. Moreover, the percentage of type I and type III collagen areas in the DPEMF and NPEMF rats was markedly lower, compared to the puncture-only rats (P < 0.001, DPEMF rats; P < 0.001, NPEMF rats). In addition, the percentage of type I and type III collagen area was remarkably elevated in the NPEMF rats, relative to the DPEMF rats (P < 0.01, Figure 3(e)). Furthermore, SEM (Figures 4(a)–4(h)) was used to observe the structural changes within the fibers of the AF-NP boundary. We revealed, using high magnification (5000x), that the fibers in the AF-NP boundary were densely and orderly arranged in sham rats. In puncture rats, however, the fibrous structure was sparse and honeycombed. Both PEMF groups showed less disorganization than the puncture group, and morphological alterations in the DPEMF rats were more organized, compared to the NPEMF rats. Taken together, these findings confirm the beneficial effect of PEMF on IVDD, with a particularly profound effect achieved by DPEMF.

The mRNA and protein expressions of collagen II, Aggrecan, and MMP-3 were examined via RT-PCR and Western blotting at 8 weeks after surgery. As depicted in Figure 5(a), the mRNA levels of collagen II in the DPEMF rats were markedly elevated, compared to the puncture and NPEMF rats, respectively (P < 0.01, DPEMF vs. puncture group; P < 0.05, DPEMF vs. NPEMF group). The mRNA expression of Aggrecan decreased significantly in the puncture only group, with respect to the sham group. PEMF treatment slightly upregulated Aggrecan expression, but no significance was achieved (Figure 5(b)). In addition, the mRNA level of MMP-3 was highest in the puncture group, followed by the NPEMF group and then the DPEMF and sham groups (Figure 5(c); P < 0.001, DPEMF vs. puncture; P < 0.05, DPEMF vs. NPEMF). The MMP-3 and collagen II protein expressions were also examined in this study. The MMP-3 protein expression in the PEMF groups was drastically reduced, compared to that in the puncture group (Figure 5(e); P < 0.01, DPEMF vs. puncture; P < 0.05, NPEMF vs. puncture), with a much lower level in the DPEMF group (Figure 5(e); P < 0.05, DPEMF vs. NPEMF). However, the collagen II protein expression in the PEMF groups was markedly elevated, relative to the puncture group (Figure 5(f); P < 0.001, DPEMF vs. puncture; P < 0.01, NPEMF vs. puncture), with a much higher level in the DPEMF group (Figure 5(f); P < 0.05, DPEMF vs. NPEMF). These collective findings indicate that PEMF strongly modulates the expression of ECM-related genes, and DPEMF has a more pronounced effect, relative to NPEMF.

Since NP cells play a crucial role in IVDD, we next explored the potential impact of PEMF on NP cells. The apoptosis rate was detected via flow cytometry at 24 h, 48 h, and 72 h after 4 h PEMF treatment. Based on our analysis, the apoptosis rate of PEMF-treated NP cells at 24 h, 48 h, and 72 h time points was similar to that of the cells that received no PEMF stimulation (Figures 6(a)–6(d)), indicating that PEMF stimulation is safe for NP cells. Next, we performed serum shock to imitate the circadian gene oscillation model in vitro. As expected, we observed circadian gene oscillation in the NP cells that received serum shock and no oscillation in the untreated cells (Figures 6(e) and 6(f)). Next, PEMF was applied to the NP cells at the Zeitgeber Times ZT8 and ZT20 (ZT0 was the start time of serum shock), which represented two typical time points that produced peaks and troughs in the cycle of the circadian clock gene oscillation. As shown in Figure 7, PEMF at different time points showed remarkably different results. PEMF at ZT8 demonstrated a strong protective effect, as evidenced by the elevated levels of ECM component protein COL2 and lower levels of ECM degeneration-related protein MMP-3, demonstrated via WB and immunofluorescence (Figures 7(a)–7(g)). However, PEMF at ZT20 demonstrated no difference in ECM-related gene expression, compared to the serum shock group. The above results indicate that the PEMF treatment displays a diurnal variation in NP cells; thus, clock genes are involved in the chronotherapy-mediation regulation of PEMF treatment.

Discs in the DPEMF and NPEMF rats were next subjected to transcriptome sequencing to identify the potential mechanism underlying the differential effect of CR and PEMF cotherapies. Based on the volcano maps, 71 differentially expressed genes (DEGs) were upregulated and 36 were downregulated between the DPEMF and NPEMF groups (Figure 8(b)). The DEG heat map between DPEMF and NPEMF groups is presented in Figure 8(a). To better understand the potential DEG functions between the two treatment groups, GO enrichment and KEGG analysis were performed to unveil mechanism(s) regulated by chronotherapy and PEMF. The top 10 GO term enrichments between the DPEMF and NPEMF groups in different categories are illustrated in Figure 8(c). Based on our data, different cellular components, biological processes, and molecular functions were enriched in the DPEMF and NPEMF groups. For instance, in molecular functions, differential genes of antioxidant activity were enriched, which indicated differences in antioxidant stress between DPEMF and NPEMF groups. Moreover, in vivo results confirmed that DPEMF upregulated SOD activity compared with NPEMF and displayed a lower MDA level. Circadian rhythm-related genes were also enriched, suggesting a possible role in PEMF treatment. The mRNA levels of four key clock genes Bmal1, Cry1, Per2, and Rev-verα were verified by RT-qPCR. The expression of clock genes Bmal1, Cry1, and Per2 but not Rev-verα was significantly elevated in the DPEMF rats, compared to the NPEMF rats (P < 0.01; P < 0.05; P < 0.05). Further KEGG pathway analysis unveiled that DEGs between DPEMF and NPEMF were mainly involved in the PI3K-AKT signaling pathway, ECM-receptor interaction, cGMP-PKG signaling pathway. Collectively, the above results indicate that circadian rhythm-related genes in IVD tissues may be a potential factor influencing the differential therapeutics of PEMF in daytime and nighttime, and the PI3K-Akt signaling pathway may be the key signaling leading to the treatment differences.