DEVELOPING NEW METHODS OF SPINAL CORD INJURY TREATMENT USING MAGNETIC NANOPARTICLES IN COMBINATION WITH ELECTROMAGNETIC FIELD DESENVOLVIMENTO DE NOVOS MÉTODOS DE TRATAMENTO DE LESÕES MEDULARES QUE USAM NANOPARTÍCULAS MAGNÉTICAS EM COMBINAÇÃO COM CAMPOS ELETROMAGNÉTICOS DESARROLLO DE NUEVOS ME

Objective: To determine the amount of loss of function after spinal cord transection of varying extents, and whether magnetic iron oxide nanoparticles, in combination with an external magnetic field, improve the rate of subsequent functional recovery in rats. Methods: The animals were divided into groups with 50%, 80% and complete spinal cord transection. The animals of all three study groups were administered magnetic iron oxide nanoparticle suspension to the area of injury. The three control groups were not administered magnetic nanoparticles, but had corresponding transection levels. All animals were exposed to a magnetic field for 4 weeks. Loss of postoperative function and subsequent recovery were assessed using the BBB motor function scale and somatosensory evoked potential monitoring on the first day after surgery, and then weekly. Terminal histological analysis was also conducted in all the groups. Results: The animals in the control or complete transection groups did not demonstrate statistically significant improvement in either the BBB scores or evoked potential amplitude over the four-week period. In the group with 50% transection, however, a statistically significant increase in evoked potential amplitude and BBB scores was observed four weeks after surgery, with the highest increase during the second week of the study. In the group with 80% transection, only improvement in evoked potential amplitude was statistically significant, although less pronounced than in the 50% transection group. Conclusion: The use of magnetic iron oxide nanoparticles in combination with a magnetic field leads to higher rates of functional recovery after spinal cord injury in laboratory animals. The mechanism of this functional improvement needs further investigation.


INTRODUCTION
Spinal cord injury (SCI) is currently one of the most serious medical problems with no effective clinical solution.It affects up to 2.5 million people worldwide, with almost 130,000 injuries a year. 1 In most cases, SCI leads to severe disability, including loss of motor and sensory function, and loss of bladder, bowel and respiratory function, depending on the level of the injury.Despite considerable progress in patient care and rehabilitation techniques, the need to develop neuroregenerative strategies for patients with SCI is indisputable.
Contrary to the peripheral nervous system, the capacity of the neural cells in adult mammalian CNS to regenerate is extremely limited.Substantial axonal regrowth in the spinal cord is only possible in the early stages of development. 2,3At later stages in life, however, the extension of axons across the injury site in the central nervous system is impeded by the formation of a glial scar that stabilizes the integrity of the damaged tissues, preventing further inflammation and possible infection.However, this process creates an insurmountable barrier for the regenerating axons, preventing the extension of their growth cones, both physically and chemically. 4,5][8][9][10][11][12][13][14] However, it is widely accepted that besides biochemical cues, the process of growth cone elongation is also influenced by tensile forces.6][17][18] The use of magnetic nanoparticles to restore loss of function related to spinal cord injury represents a promising approach to this challenge, as it allows spatial modulation of the axonal elongation process, without inducing further damage to the injured CNS tissue.
0][21][22][23] Their small size, high biocompatibility, readily available protocols of surface functionalization with various biological molecules and, most importantly, the ability to interact with magnetic field forces, meet the requirements needed for potential mechanical stimulation of axonal growth.
The current experimental study aims to determine the effectiveness of magnetic iron oxide nanoparticles in functional recovery from SCI through potential stimulation of in vivo axon growth and elongation in the presence of an external magnetic force.

METHODS
This research was approved by the N.N.Priorov Central Institute of Traumatology and Orthopedics Ethics Committee (#12548977).

Experimental design
The study was conducted on 72 healthy adult (8-10 weeks) female Wistar rats, weighing 220-310 g, which were kept at 23°C on a regular 12 h light/dark cycle with a carefully maintained diet.The animals were divided into 6 groups, according to the extent of spinal cord transection.The rats underwent a 50% spinal cord transection in the first study group, an 80% transection in the second group, and a complete transection in the third study group.All the animals in the three study groups were administered a magnetic iron oxide nanoparticle (Fe 3 O 4 , 10-50 nm in diameter) suspension to the area of injury, through a polyethylene catheter, every other day for 4 weeks.The other three groups were control groups, with no magnetic nanoparticles administered but with corresponding transection levels.All the animals were exposed to a static direct current 3 mT magnetic field for 5 hours, every other day, for 4 weeks.

Surgical procedures
All the surgeries were conducted by the same surgeons, using a surgical microscope, under strict aseptic conditions, maintaining the animals' body temperature at 38°C.The animals were anesthetized intraperitoneally using xylazine (10 mg/kg) and ketamine (100 mg/ kg).Additional doses were administered intraoperatively, as needed.
The thoracic area was shaved and cleaned with iodine solution and 70% alcohol.After a 4 cm midline incision and paraspinal muscle separation, a laminectomy was performed at the T9/T10 level using microsurgical rongeurs to expose the spinal cord.The spinal cord was transected (50%, 80% and complete transection) with a 0.1 mm blade using a custom-made guard, taking care to cause minimal damage to the surrounding arteries.After confirming the transection, the muscles and skin were closed in layers.After surgery, 5 mL of D5 lactated Ringer's solution was injected intraperitoneally to prevent dehydration, and the animals were kept on a heating pad to maintain body temperature for two hours.During the first three days following surgery, all the animals were injected with gentamicin-cefazolin 20 mg/ kg to prevent infection, and food and water were provided ad libitum.If spontaneous urination was not observed, the urinary bladder was manually evacuated two times a day.

Behavioral and electrophysiological assessment
Spontaneous function of the hind limbs was evaluated on the first day after surgery and then weekly for 4 weeks.The scoring was done by blinded independent observers using the BBB (Basso, Beattie, and Bresnahan) motor function scale. 24The BBB scale allows for quantitative analysis of sensorimotor function after SCI, and ranges from zero (no movement) to 21 (normal gait with full coordination) points.The scoring is based on the assessment of joint movement, paw placement and gait stability.Scores ranging from 0 to 7 points mainly describe the movements in the large joints of the hind limbs, scores of 8 to 13 describe coordination and paw placement, and scores of 14 and higher describe overall stability of the gait.
Electrophysiological evaluation of the functional integrity of the descending tracts of the spinal cord was conducted before surgery to determine the baseline values, then on the first day after surgery, and weekly for 4 weeks thereafter.The animals were anesthetized (with xylazine and ketamine, as described above) and evaluated using compound motor evoked potential (MEP) testing. 25The potentials were recorded from the tibialis anterior muscles of the hind limbs after transcranial stimulation of the brain cortex using bipolar needle electrodes.The duration of the stimulus was 0.2 ms and its intensity was 10 mA at 10 Hz.A minimum of three MEP traces were recorded over a 25 ms duration.The recorded traces were amplified and digitized.The amplitude was measured in μV.

Histological analysis
After four weeks, the animals were deeply anesthetized by intraperitoneal injection of ketamine (70 mg/kg) and perfused transcardially with cold saline solution followed by 4% paraformaldehyde in phosphate-buffered saline 0.1 M. The vertebral column was extracted from T7 to L3 and post-fixed according to the standard protocol.A spinal cord tissue block that included the region of transection was then cryosectioned, stained (H&E, Masson's trichrome), and mounted on a slide.The stained spinal cord lesion sites were then analyzed under a microscope (Nikon Corporation, Japan) using image analysis software.

Statistical analysis
Statistical analysis was performed using Statistical Package for Social Sciences version 18 software (SPSS Inc, Chicago, IL, USA).Data comparison between the groups was performed for each weekly time point using the Kruskal-Wallis test, with post hoc analysis using the Mann-Whitney U test.All results were expressed as means ± standard error of the mean (SEM).The criterion level for statistical significance was set at a P value of less than 0.05.

Postsurgical survival rate
The survival rate after spinal cord transection was 85% (61 out of 72 animals) on the day after surgery.By the end of the first week, 51 animals (71%) survived, and remained in the experiment for the remainder of the four weeks.Animals with complete spinal cord transection were less likely to survive, which was most likely due to self-mutilation and urinary bladder rupture or urinary tract infection.The final data was collected for the following number of animals in each group: 50% transection + MNP -10 (out of 12); 80% + MNP -11 (out of 12); 100% transection + MNP -7 (out of 12); 50% control -9 (out of 12); 80% control -8 (out of 12); 100% control -6 (out of 12).

Behavioral assessment results
As expected, the animals in all the groups showed a drastic decrease in function on the first day after surgery, with no or minimal voluntary movement observed in the hind limbs.On the first day after transection, the BBB scores decreased from an average of 21.0±0 points at the pre-injury baseline to an average of 1.1±0.8points.There were no statistically significant functional differences between groups on the first day after surgery.One week after surgery, the animals in the control groups did not demonstrate statistically significant improvement in BBB scores or evoked potential amplitude over the four-week period.Neither was any statistically significant improvement observed in the study group with complete spinal cord transection.In the group with 50% transection, however, there was an average of 4.3±1.4point increase (p<0.001) in BBB scores four weeks after surgery, with slight or extensive movement in two or more joints in the hind limbs, albeit with no or very little weight bearing.In the group with 80% spinal cord transection, the average increase after 4 weeks was 2.3±1.7 points (p=0.061), which was close to the statistical significance threshold.The BBB scores increased gradually over the four-week period, with the most marked improvement being observed during the second week of the study.(Figure 1)

Electrophysiological assessment results
As with the functional scores, on the first day after surgery, the average amplitudes of the evoked potentials demonstrated a drastic decrease in all groups, with failure to register any potentials in some animals.The differences in amplitude between groups on the first day after surgery were not statistically significant, although the average amplitudes in the 50%-transection groups were slightly higher.On the first postoperative day, the average amplitudes constituted on average 4.7±1.4% of the preoperative amplitude values (average of 538.7±91.5 μV).
The animals in the control groups or the study group with complete spinal cord transection also did not demonstrate statistically significant increase in evoked potential amplitude over the four-week period.The group with 50% transection demonstrated gradual improvement in electrophysiological response, with the average amplitude reaching 14.1±3.5% (p<0.001) of the preoperative value by the end of the fourth week.There was also a statistically significant improvement in amplitude in the group with 80% spinal cord transection four weeks after surgery, which constituted 5.0±3.0%(p<0.05) of the original value.In both cases, the most rapid increase in amplitude was observed during the second week of the study.(Figure 2) Repeated transection of the spinal cord rostral to the original lesion site abolished the recovered hind limb movements and evoked potential improvements in both groups.

Histopathological assessment results
Histological analysis of the lesion sites performed 4 weeks after surgery yielded no significant differences in terms of hemorrhage, necrosis or cellular infiltration.However, the experimental groups had higher viable motor neuron counts and thicker myelinated fibers.Measurement of the cavity area showed an area that was, on average, 17.8±2.4% times smaller (p<0.05) in the experimental group with 50% transection.Although Masson's trichrome staining demonstrated pronounced glial reactions in the areas next to the lesions, collagen content was significantly lower in the experimental group with 50% transection.

Figure 2 .
Figure 2. Comparison of the changes in evoked potential amplitudes over 4 weeks.(MNP -magnetic nanoparticles).