Tumor-Specific Alterations in Motor Cortex Excitability and Tractography of the Corticospinal Tract—A Navigated Transcranial Magnetic Stimulation Study

Background : Non-invasive brain mapping using navigated transcranial magnetic stimulation (nTMS) is a valuable tool prior to resection of malignant brain tumors. With nTMS motor mapping, it is additionally possible to analyze the function of the motor system and to evaluate tumor-induced neuroplasticity. Distinct changes in motor cortex excitability induced by certain malignant brain tumors are a focal point of research. Methods : A retrospective single-center study was conducted involving patients with malignant brain tumors. Clinical data, resting motor threshold (rMT), and nTMS-based tractography were evaluated. The interhemispheric rMT-ratio (rMT Tumor /rMT Control ) was calculated for each extremity and considered pathological if it was > 110% or < 90%. Distances between the corticospinal tract and the tumor (lesion-to-tract-distance – LTD) were measured. Results : 49 patients were evaluated. 16 patients (32.7%) had a preoperative motor deficit. The cohort comprised 22 glioblastomas (44.9%), 5 gliomas of Classification of Tumors of the Central Nervous System (CNS WHO) grade 3 (10.2%), 6 gliomas of CNS WHO grade 2 (12.2%) and 16 cerebral metastases (32.7%). 26 (53.1%) had a pathological rMT-ratio for the upper extremity and 35 (71.4%) for the lower extremity. All patients with tumor-induced motor deficits had pathological interhemispheric rMT-ratios, and presence of tumor-induced motor deficits was associated with infiltration of the tumor to the nTMS-positive cortex ( p = 0.04) and shorter LTDs (all p < 0.021). Pathological interhemispheric rMT-ratio for the upper extremity was associated with cerebral metastases, but not with gliomas ( p = 0.002). Conclusions : Our study underlines the diagnostic potential of nTMS motor mapping to go beyond surgical risk stratification. Pathological alterations in motor cortex excitability can be measured with nTMS mapping. Pathological cortical excitability was more frequent in cerebral metastases than in gliomas.

Analysis of both cortical and subcortical motor areas is correlated with postoperative outcome after resection of the lesion.This makes nTMS-based DTI-FT a diagnostic and prognostic tool in neurosurgery [8,9,21,22,26,32,33]. Key parameters of nTMS-based risk assessment are the resting motor threshold (rMT) and the interhemispheric rMT-ratio of the tumorous and the non-affected hemisphere [8,9,32].This ratio is considered pathological if it exceeds 110% or falls below 90%.A pathological interhemispheric rMT-ratio is associated with an increased risk of permanent surgery-related motor deficits [8].Furthermore, the distance between the lesion and the CST (lesion-totract-distance -LTD) correlates inversely with the risk for surgery-related paresis [8,9,21,32,34].
Apart from risk stratification and preoperative visualization of motor cortex and motor pathways, nTMS mapping serves as a tool to evaluate motor function and motor cortex reorganization in brain tumor patients [6,[35][36][37][38][39][40][41][42][43].nTMS data correspond with tumor grading and motor status: high grade gliomas are associated with higher rates of pathological rMT-ratios of upper and lower limb muscles compared with lower grade gliomas, indicating a measurable distraction of the motor system induced by more aggressive tumors [37][38][39].Additionally, edema, intake of anticonvulsive medication, and tumor location significantly influence the rMT of the tumorous hemisphere [36,43].To the best of our knowledge, there are no data regarding the influence of different tumor entities, such as low grade gliomas, high grade gliomas and cerebral metastases, on cortical excitability and interhemispheric motor threshold ratio using nTMS.Consequently, we conducted this study to add further information to this phenomenon of alterations in cortical excitability and nTMS-based-DTI-fibertracking in patients with brain tumors.

Ethical Considerations
The study design was approved by the institutional review board of Paracelsus Medical University Nuremberg (IRB-2024-10).The study was conducted in accordance with the Declaration of Helsinki.Informed consent was waived due to the retrospective study design.

Study Design and Inclusion Criteria
Patients that were treated surgically between July 2021 and March 2024 for histologically confirmed glioma or metastases and received navigated transcranial magnetic stimulation for mapping the motor cortex were included into this retrospective study.Inclusion criteria were age over 18 years and presence of one single supratentorial lesion.Patients with multiple lesions or infratentorial lesions were excluded from the analysis.Patients with motor deficits that were related to a previous surgery were excluded.

Clinical Data
Apart from demographic data, intake of anticonvulsive medication, tumor histology, and the presence of motor deficits were registered.All motor deficits were considered to be induced by the tumor.

Navigated Brain Stimulation Protocol
Bilateral nTMS motor mapping using the Nexstim NBS 5 system (Nexstim Oy, Helsinki, Finland) was performed in each patient as part of the preoperative diagnostic workflow.All examinations were conducted by one investigator (T.E.).The workflow was in accordance with published consensus guidelines [44]; however, some modifications were made to the mapping procedure.These are outlined in the following.
The patient's head was co-registered with the magnetic resonance (MR) reference exam.Pre-glued surface electrodes (Ambu Neuroline 700, Ambu, Ballerup, Denmark) were attached to the target muscles (abductor pollicis brevis (APB), abductor digiti minimi (ADM), one forearm muscle, and the tibialis anterior (TA) and gastrocne-mius muscle (GC)) in a belly-tendon-fashion.The ground electrode was placed at the patient's elbow.In all examinations, the non-affected hemisphere was mapped first.The first step of the mapping procedure was to determine the hand motor hotspot within the primary motor cortex.The spot which elicited the greatest motor evoked potential (MEP) amplitudes during the first round of mapping was considered as the motor hotspot.Stimulations were repeated at that spot to confirm the location of the hotspot.The next step was the determination of the rMT using the NBS 5's inbuilt motor threshold hunting algorithm.The stimulation intensity which elicited MEPs with amplitudes >50 µV in >50% of all applied stimulations was considered the rMT of that hemisphere.The upper extremity muscles were subsequently mapped at 110% of the patientindividual upper extremity rMT.The lower extremity was mapped in the same fashion: after determining the rMT for the lower extremity, the cortical representations of the lower limb were mapped at 110% of the patient-individual lower extremity rMT.The coil orientation was perpendicular to the central sulcus following the shape of the precentral gyrus during mapping of the primary motor cortex of the upper extremity and rotated perpendicular to the sinus sagittalis superior for mapping the lower extremity muscles.
All examinations were evaluated post-hoc and motor positive spots (MEPs with a peak-to-peak amplitude >50 µV and latency between 10 ms and 30 ms) were exported via the digital imaging and communications in medicine (DICOM) format for nTMS-based DTI fibertracking.Fig. 1 illustrates the visualization of the motor cortex.

Imaging and Tractography
All patients received contrast enhanced MR-imaging, with a voxel size of 1 × 1 × 1 mm 3 , and diffusion tensor imaging (DTI with 32 gradient directions, voxel size 2 × 2 × 2 mm 3 ).Tumor and edema volumes were measured using a Medtronic® StealthStation™ S8 (Medtronic Inc., Louisville, CO, USA) and a semiautomatic segmentation algorithm.For enhancing tumors, the T1 post-contrast sequence was used for volumetric assessment, whereas T2/fluid attenuated inversion recovery (FLAIR) sequences were used for non-enhancing tumors.Edema volumes were evaluated using the T2/FLAIR sequences in all patients.Homogenous T2 hyperintense non-enhancing gliomas were defined as having no vasogenic edema.
All nTMS motor positive spots were imported into the Medtronic® Stealthviz™ software (Medtronic Inc., Louisville, CO, USA), enlarged to a diameter of 6 mm per spot and defined as a cortical region of interest.A second region of interest was placed into the caudal pons.CSTs of both hemispheres were visualized at 75%, 50% Categorial variables were tested with the Fisher Exact Test.Continuous variables were tested for significance using the Mann-Whitney-U-Test.LTD, lesion to tract distance; FAT, fractional anisotropy threshold.
and 25% of the patient's individual fractional anisotropy threshold (FAT, i.e., the highest fractional anisotropy to visualize fibers belonging to the corticospinal tract; 75%FAT, 50%FAT, 25%FAT) at a directional angle of 60°(upper extremity) or 45°(lower extremity) and a minimum fiber length of 110 mm.The minimum distance between the CST and the tumor (LTD) was measured for each fractional anisotropy (FA)-value.For distinct analyses, the LTDs were measured for upper and lower limb CST-fibers separately.

Data Analysis
The data analysis was subdivided into two parts.
(1) Influence of motor status and tumor subtype on cortical excitability measured with rMT and interhemispheric rMT-ratio.
(2) Influence of motor status and tumor subtype on CST reconstructions measured in FAT and LTD.
IBM SPSS Version 29.0 (IBM Corp., Chicago, IL, USA) for Microsoft Windows was used for statistical analysis.Metric variables are shown as means ± standard deviation (SD), and categorial variables as absolute number (n) and percentage (%).Categorial variables were tested using Fisher Exact Tests for dichotomous variables and Fisher-Freeman-Halton-Tests for categorial variables with more than two categories (e.g., histology or tumor location).For statistical testing of continuous variables, Mann-Whitney-U-Tests and Kruskal-Wallis-Tests were used.Multivariate analyses were carried out for possible confounders for a pathological interhemispheric rMT-ratio.A backward elimination logistic regression model was used.Nagelkerke's R 2 , regression coefficient B, odds ratio (OR), and 95% confidence intervals (95% CI) were also calculated.
A p-value < 0.05 was considered statistically significant.

nTMS Data
nTMS was successfully conducted in all patients of the cohort.Motor evoked potentials could be elicited from each monitored muscle.Mean rMT Tumor for the upper extremity was 33.2 ± 7.3% stimulator output, whereas the rMT Tumor for the lower extremity was 53.9 ± 13.4% stimulator output.26 patients (53.1%) had a pathological rMTratio for the upper extremity and 35 (71.4%) had a pathological rMT-ratio for the lower extremity muscles.12 patients (24.5%) had nTMS-positive spots at the tumor margins and rMT Tumor for the upper extremity tended to be higher in these cases (p = 0.09), whereas the interhemispheric rMTratios were not pathological (p = 0.33 for the upper extremity and p = 0.47 for the lower extremity, respectively).However, nTMS positive spots at the tumor margin were associated with a tumor-induced motor deficit (p = 0.04).
Patients with motor deficits did not exhibit altered rMT-values and interhemispheric rMT-ratios only tended towards significance in these patients.No patient with a tumor-induced motor deficit had normal interhemispheric rMT-ratios for both the upper and the lower extremities (Table 2).We did not discover differences in rMT-values for any extremities in patients with gliomas or metastases (Fig. 2, Table 3).In contrast, patients with cerebral metastases were found to have higher rates of pathological rMT-ratios for the upper extremity muscles compared with the other tumor entities of the patient sample (p = 0.002).In the binary logistic regression model, which comprised age, sex, edema volume, LTD, and histological diagnosis, cerebral metastasis remained the only significant variable (OR = 21.67,95% CI = 1.8-260.57,p = 0.015, Nagelkerke's R 2 = 0.38, p < 0.001 for the regression model, Table 4).
In the subgroup of glioma patients, pathological rMTratios for the upper extremity were more frequent in glioblastomas compared with grade 2 and 3 tumors (p = 0.04).
The results of the nTMS examinations are shown in Tables 2,3,4.

nTMS-Based DTI-Fibertracking
In the tractography analysis, we observed lower LTDs if patients had motoric deficits, whereas FATs did not differ (Fig. 3).Regarding the different histological tumor types, there were no alterations in FATs between gliomas or metastases and no differences in involvement of the corticospinal tract (Fig. 3B, Table 3).
Taking the results of the nTMS examinations into account, there were no differences in upper-extremity LTDs, if the interhemispheric rMT-ratio for the upper extremity was pathological (Fig. 4A), whereas LTDs were significantly shorter for the lower limb CST if the lower extremity had a pathological interhemispheric rMT-ratio (Fig. 4B).LTD measurements only tended towards shorter distances if patients developed new motoric deficits postoperatively (p = 0.16 for 75%FAT, p = 0.096 for 50%FAT, and p = 0.11 for 25%FAT for worsening in motor function at discharge; and all p > 0.09 for persistent deficit at 3 months).13 patients showed worsening in motor function at discharge and the deficits were transient in 7 of these patients.Two patients were lost to follow-up.

Discussion
The aim of this study was to analyze the cortical excitability among patients with and without tumor-induced motoric deficits and to further differentiate the nTMS-and fibertracking-derived characteristics of different malignant brain tumors.In our cohort, all patients with tumor-induced motor deficits had abnormal interhemispheric rMT-ratios of the upper or lower limb.Additionally, infiltration of the nTMSpositive cortex was associated with tumor-induced motor symptoms as a proof of concept of nTMS.Regarding tumor histology, patients with cerebral metastases were more likely to have a pathological rMT-ratio of the upper extremity compared with glioma patients.The results of the tractography analyses confirmed the closer spatial relation between motor eloquent brain and motor symptoms, and LTD were shorter in patients with pathological cortical excitability.
The results of our study partly support the results published by Lavrador and Mirchandani et al. [37][38][39].In their study of intrinsic brain tumors, higher tumor grading is associated with pathological cortical excitability, especially for the lower limb muscles, whereas we found this result for the upper but not for the lower extremity.Additionally, we found alterations in metastases compared with gliomas, which has not been shown before.Furthermore, tractography reveals changes in CST diffusion parameters in glioma patients compared with healthy participants [39].The pathological changes of the CST correlated well with the nTMS-parameters in their study [39].Using nTMS  Glioma patients had altered excitability compared with patients with cerebral metastases indicating different impact on the motor system by different tumors, whereas the spatial distance between the lesion and the motor pathways was not different among the tumor entities in our co-hort.We hypothesize that there are different neuroplastic compensation mechanisms in gliomas compared with cerebral metastases in order to counterbalance the disruption of the motor system by an intrinsic brain tumor.Neuroplasticity is well-described by nTMS in glioma patients [40][41][42]45].
Previously published studies found associations of motor status, tumor histology, and altered cortical excitability [6,36,43].Higher motor thresholds are associated with motoric deficits and an interhemispheric rMT-ratio of >110% is associated with imminent and irreversible disruption of the motor system [8].In the study conducted by Sollmann et al. [21], motor thresholds correlated inversely with the distance between the CST and the tumor in patients presenting postoperative deficit.Despite predicting postoperative motor function, the association of tumorinduced motor deficits and tractography results remains unclear.Sollmann et al. [21] found partially significant results regarding LTD and preoperative paresis, whereas the LTDs in our study were significantly shorter if patients had motoric deficits, and LTDs were shorter in patients with pathological rMT-ratios of the lower extremity.Several studies report increased risk for surgery-induced paresis in cases with close LTDs [8,9,21,26,33,46].The results of these risk stratification analyses indicate a tight relationship between LTD and postoperative paresis.Similarly to the surgery-related paresis, we were able show the spatial relationship for tumor-induced (i.e., pre-treatment) motor deficits in our cohort.Patients with motoric deficits had significantly shorter LTDs for all CST-reconstructions and, furthermore, the nTMS-positive cortex was infiltrated by the tumor in patients with a motor deficit.This relationship additionally serves as a proof of concept of nTMS as a valuable and reliable tool for non-invasive motor mapping and nTMS-based tractography.

Strengths and Limitations
The results of our study are limited by the retrospective study design and the small sample size.It was not possible to further differentiate between the intensity of motor symptoms.In contrast, the nTMS workflow and nTMSbased fibertracking followed a strict and standardized protocol and all examinations were conducted by the same examiner, ensuring the highest possible standards and comparability.Additional limitations arise from the fact that DTI tractography is based upon software algorithms with known limitations in voxels of crossing fibers and areas of extensive edema [47,48].Taking the limitations of DTI tractography into account, we might have underestimated the impact of altered nTMS-parameters and pathological rMT-ratios on the results of tractography.

Conclusions
The results of our study indicate altered cortical excitability in patients with gliomas and metastases.Cerebral metastases were associated with abnormal rMT-ratio of the upper extremity, whereas the cortical excitability was only partly associated with the tractography analysis.Tractography analyses further correlated well with the motor status of the patients.

Fig. 1 .
Fig. 1.Visualization of the motor cortex.Orange spots indicate motor-positive spots.The red-blue double arrow indicates the position and orientation of the stimulation coil.

Fig. 2 .
Fig. 2. Pathological interhemispheric rMT-ratios depending on tumor histology.(A) Patients with cerebral metastases had higher rates of pathological interhemispheric rMT-ratios of the upper extremity compared with glioma patients (p = 0.002).(B) The distribution of pathological interhemispheric rMT-ratios for the lower extremity muscles was not significantly different among different tumor entities (p = 0.36).Fisher-Freeman-Halton-Test.rMT, resting motor threshold.

Fig. 3 .
Fig. 3. Comparison of fibertracking results according to motor status and tumor entity.(A) FATs showed no difference in patients with tumor-induced motor deficits (all p > 0.065).(B) FATs showed no difference among tumor entities (all p > 0.22).(C) LTDs were shorter if patients had a preoperative motor deficit (p = 0.020 for 75%FAT, p = 0.021 for 50%FAT, and p = 0.012 for 25%FAT).The closest LTD (for either upper or lower CST fibers) is analyzed here.Table 2 shows LTD measurements separated for upper and lower limb CST fibers.Boxes are based on medians and interquartile ranges.Significant results are marked with * for p < 0.05, Mann-Whitney-U-Tests and Kruskal-Wallis Tests.

Fig. 4 .
Fig. 4. Comparison of LTD measurements in patients with and without a pathological interhemispheric rMT-ratio.(A) LTDs for the upper limb showed no difference if rMT-ratios were pathological for the upper extremity.(all p > 0.17).(B) LTDs for the lower limb were shorter if rMT-ratios were pathological for the lower extremity muscles (p = 0.02 for 75%FAT, p = 0.005 for 50%FAT and p = 0.005 for 25%FAT).Boxes are based on medians and interquartile ranges.Significant results are marked with * for p < 0.05 and ** for p < 0.01, Mann-Whitney-U-Tests.

Table 1 . Baseline data of the patient sample.
SD, standard deviation; N, number; CNS, central nervous system; nTMS, navigated transcranial magnetic stimulation; rMT, resting motor threshold.

Table 3 . Comparison of patients according to tumor histology.
Categorial variables were tested with the Fisher-Freeman-Halton-Test.Continuous variables were tested for significance using the Kruskal-Wallis-Test.

Table 4 . Results of the binary logistic regression model to predict a pathological interhemispheric rMT-ratio for the upper extremity.
Logistic regression backward elimination model (final model, step 5: Nagelkerke R 2 = 0.38, p < 0.001)