Peripheral nerve injury produces loss of sensory and motor function, resulting in critical economic and psychological issues and diminished quality of life. Common causes of peripheral nerve injuries are traffic accidents, firearm injuries, chemical injuries, cutting tool injuries, and crushing injuries [1]. Nerve injuries may also result from dental procedures, such as extraction, implantation, minor surgery, and root canal treatment (chemical injury), and trauma [2]. There is no doubt that an accurate diagnosis leads to accurate treatment planning. However, these diagnoses are based on the patient's subjective symptoms. An objective and accurate diagnosis can help achieve successful treatment outcomes. To our knowledge, this is the first study to evaluate peripheral nerve injury according to the severity of damage using 18F-FDG PET/MRI in a rat model of sciatic nerve injury.
The nerve damage models were divided into three groups according to the crushing time. Alvites et al. reported that in most studies, the crushing time was different for each paper [12]. There was no standardized protocol for crushing time, which ranged from 15 seconds to two hours [12-19]. An et al. attempted to objectify the damage intensity by varying the number of notches on the forceps and the pinching part of forceps to damage nerves [20]. This study attempted to examine the differences in sciatic nerve damage according to the crushing time.
Other papers have used various crushing load tools, such as Jeweler’s forceps, Kocher's forceps, pincers, mosquito forceps, and aneurysmal clips [12]. Serrated and non-serrated forceps have also been used without distinction. In this experiment, a curved serrated hemostat was used; however, the serration was in one specific direction, and a crushing injury could be applied only to a specific part of the nerve by clamping one time. Thus, there may have been part of the nerve where a crushing injury was not applied. Thus, it was difficult to inflict more than a certain level of injury. Beer et al. used a non-serrated clamp to standardize their nerve crush model to ensure that the pressure on the injury site was uniformly transmitted [21]. Future studies should develop a consistent crushing injury model using tools, such as non-serrated hemostats, generating a crushing injury by turning the tool 90 degrees after crushing once, or causing a crushing injury by changing the angle of two hemostats by 90 degrees.
There were several limitations in measuring the PWT with von Frey filament. One limitation of the von Frey filament test was that it did not allow for differentiation between a pain response and routine bodily grooming/itching. Mason et al. stated that it was important to note the duration of a specific behavior to differentiate between pain and grooming. Typically, a pain response is one swipe after filament application; however, a grooming motion tends to be longer and can last from a few seconds to a few minutes. If the grooming/itching behavior is indistinguishable due to irritability, it is best not to record it as a positive response [22]. When a manual filament was used, there was a possibility that the experimenter may have had a subjective interpretation. Therefore, both hind paws were measured three times, and the mean value was used for analysis.
When changing from one filament to the next, the increase in force was not linear, and the force gap between filaments was very large. For example, after using a 60 g filament, the next filament force was 100 g, followed by 180 g and 300 g. As the force increased, the filament thickened, and it was not possible to obtain accurate measurements for force alone. Therefore, the pressure, which is the force per unit area of the filament, was calculated. The results showed no significant difference between force and pressure. However, future studies should use pressure units instead force to obtain more precise data. In light of these points, the PWT test showed subjective results similar to those of neurosensory testing in clinical practice.
Nevertheless, the withdrawal threshold result was consistent with the results of other research. Roman et al. demonstrated hypoesthesia by time in the first, second, and third weeks; the peak was in the first and second weeks, and hypoesthesia gradually improved and recovered by the third week [17]. The histological findings were also consistent with those of other studies. In a sciatic nerve injury model by Zhang et al., the IHC findings showed Schwann cell proliferation over time, with a peak in the first and second weeks, followed by a decrease in the third week [23].
Histological analysis was conducted using the IHC method. Schwann cells play vital roles in peripheral nerve regeneration [24]. However, identification by conventional histological methods is difficult. Schwann cells can be identified using an antibody against the S-100 protein. Detection of Schwann cells by IHC staining is considered to be a positive indicator of nerve regeneration [25]. Furthermore, accumulation of S-100 protein indicates the proliferation of sciatic nerve Schwann cells. Proliferating Schwann cells promote the sustained regeneration and functional recovery of the sciatic nerves [26]. Zhao et al. used S-100 as a Schwann cell marker to compare the effect of decimeter wave therapy on the proliferation of Schwann cells after nerve injury [10]. S-100 may serve as a marker for the proliferation of Schwann cells in sciatic nerve regeneration research [27]. Wang et al. reported that ginsenoside Re significantly increased S-100 expression in Schwann cells to promote rat sciatic nerve regeneration [28]. In this experimental study, S-100 intensity was measured, and the values (IntR) were compared with the SUVR results.
Many studies have focused on noninvasive diagnostic tools using imaging methods such as US to evaluate nerve hypertrophy and intraneural vascularization [29, 30]. The quantity and quality of nerve damage have been studied using diffusion tensor imaging [31]. Moreover, radioactive tracers for PET scan have been used to assess the severity of nerve damage. PET alone has some advantages and disadvantages. Although it can identify nerve damage, it is difficult to determine the exact location of damage. Purohit et al. stated that researchers should be aware of several specific patterns of FDG uptake and suggested that contrast-enhanced CT, US, or MRI be used together to prevent errors in PET interpretation [3]. In particular, in this experiment, PET/MRI fusion was used to avoid the limitations of PET and utilize the advantages of MRI. This study used PET/MRI fusion to obtain metabolic 18F-FDG PET images with a more sensitive anatomical evaluation with MRI.
The results confirmed that PET imaging and IHC values increased as the nerve-crushing time increased. Both the imaging and histological results showed a significant difference between 30 seconds and five minutes of crushing injury. However, there was no significant difference between two and five minutes for both results. One reason is that with sufficient strength, a curved hemostat can produce a crushing injury even in two minutes; therefore, there was no difference between two and five minutes. To elicit a difference in time through crushing injury, it is worth considering reducing the crushing time to one minute or ratcheting the clamp a different number of times. Conversely, investigator should consider methods other than clamping for crush durations of more than two minutes. Another study reported that crushing injury through clamping could not produce more severe injuries, such as neurotmesis [20]. Therefore, no significant difference was expected between two and five minutes of crush injury.
Unlike the imaging analysis, the histological results showed no statistically significant difference between 30 seconds and two minutes. This finding could result in a subjective interpretation of histological characteristics. In addition, obtaining reliable histological data may be difficult because specialized equipment for each process is required for histological analysis [32]. Therefore, this study used computer-aided IHC analysis to minimize subjectivity in interpretation. The biggest advantage of IHC compared with immunofluorescence (IF) is that IHC staining is permanent with no change in color. Moreover, tissue morphology is clearly visible, simplifying interpretation. With IF, fluorescence eventually disappears; therefore, photographing and scans are necessary to maintain a record [33]. A disadvantage is that fluorescence can be automatically generated when using formalin-fixed paraffin-embedded tissue (FFPE) and seen in autofluorescent elements such as, collagen, elastin, neutrophils, and blood vessels. Therefore, it is very important to restore slides that are not stained with isotype controls and use frozen sections instead of FFPE tissues. An advantage of IF is that it allows immediate visualization of cell populations through various staining techniques on one slide. Recently, IF has been widely used in immune-oncology research. Fluorescence quantification can provide better visualization and more sensitive results for future experiments [34, 35].
This experiment is very important in that crush damage was imposed differently over time. This study aimed to quantify the SUVR according to the severity of injury in a rat sciatic nerve injury model using PET/MRI. When a nerve is damaged, it will be possible to objectively quantify the severity of damage using PET imaging. As a result, it will be also possible to predict how long it will take to recover if long-term data are accumulated.
There are errors and difficulties in obtaining measurements with manual filaments. However, these aspects are similar to the neurologic examinations currently performed in clinical practice, and the findings may vary depending on the measurer’s subjective interpretation and the subject’s sensory diversity, which are current limitations. With advancements in computer-aided image capture systems, non-communicating and non-stimulus-evoked pain evaluations may become more accurate and useful [36]. The sciatic nerve can be evaluated by analyzing the toe angle during the gait stance duration, ankle kinematics, and gait through video recording for motion evaluation without considering the method of evaluation by applying pressure [12]. The analysis is technically complex and necessitates appropriate equipment. However, more precise results can be obtained if this method is also used for further research.
As there were only six rats in each group, there were some differences in the values, but no statistically significant differences. Future studies should include at least 30 rats and use statistical methods with parametric tests to yield more sensitive and accurate results. Moreover, PET/MRI can be used to quantify the severity of nerve damage and predict prognosis. Thus, PET/MRI can objectively evaluate treatment efficacy when studying new drugs used for neuropathic pain. Doctors often face the challenges of defining a diagnosis and prognosis based only on the patient's subjective symptoms. Patients have difficulties describing the symptoms of their sensory numbness and may feel frustrated. However, objective tools can diagnose nerve injury, predict prognosis, and provide treatment satisfaction for doctors and patients.