Gadolinium contrast agents: dermal deposits and potential effects on epidermal small nerve fibers

Subject and samples

This prospective observational study was carried out in three German neuromuscular centers (Giessen, Ulm and Mainz). Inclusion criteria were definite SFN according to the NEURODIAB criteria [19]. Patients were included in the study if idiopathic SFN was confirmed by clinical, neurophysiological, laboratory and genetic investigations. Patients were excluded if they had clinical signs of large fiber involvement, pathological nerve conduction (NC) studies or an underlying aetiology for SFN was present. Besides metabolic causes, infectious diseases, immune-mediated and paraneoplastic syndromes, genetic syndromes such as sodium channelopathies, Fabry Disease and TTR amyloidosis were ruled out [20]. During a standardized interview, the iSFN patients were asked for GBCA exposure, how often GBCAs were applicate, and the time point of the last exposure. If the patients were unsure, the radiologist responsible for the MR examinations was contacted and type, brand and volume of GBCA applied was noted. If the GBCA exposure remained unknown the patients were excluded.

The final sample consisted of twenty-eight patients fulfilling the NEURODIAB criteria of definite SFN. Of these participants, 23 iSFN patients (82%) reported exposures to GBCAs (iSFNe) and 5 (18%) declared that they have never been exposed to GBCAs (iSFNne). These frequencies significantly (Chi2 = 11.6, p < 0.001) deviate from the expected equal distribution (14 cases/exposure group). Quantitative sensory testing (QST) could be performed in 15 patients (54%). The distribution across the GBCA exposure groups is given in Table 1. The reasons for the MRI examinations were heterogeneous including imaging of brain, joints, and pelvis. The gender and age distribution of these three groups as well as the respective values of the controls are given in Table 1.

Table 1 Demographic data and descriptive statistics for age and exposure to GBCA for iSFN patients/controls and frequencies distribution of quantitative sensory testing (QST)

All patients received a skin biopsy at the distal leg according to recommendations [8]. Additional skin biopsies from six healthy subjects without history for GBCA exposure or neuropathic pain were included in the study.

Ethics and approval

The study was approved by the central Ethics Committee of the Justus-Liebig-University of Giessen (ethics approval number AZ 27/20) as well as the local ethic committees from the participating centers. The Ethics Committees approved the conducted experiments on human participants. Informed and written consent was obtained from all participants. The study was conducted according to the current version of the Guidelines for Good Clinical Practice and Helsinki Declaration of the World Medical Association.

Quantifying intraepidermal nerve density (IENFD)

To determine the intraepidermal nerve fiber density (IENFD) standard procedures were performed. From each biopsy, sections were stained with antibody against Protein Gene Product (PGP) 9.5 a neuron-specific protein that labels axons in the peripheral nervous system [21, 22]. IENFD was determined according to published counting recommendations. For all analyses, IENFD were z-transformed \(_}=\frac}_}-}_}}}_}} )\) using the age-and sex-matched reference values. IENFD was considered significantly “reduced” when it was below the 5% percentile of the reference data (zIENFD < 1.64) [7]. The investigators were blinded to the samples during the morphometric analysis.

Elemental bioimaging of gadolinium deposits in skin samples

From each skin biopsy sample, 10 µm thick cryosections were prepared and subjected to an elemental bioimaging procedure that can detect Gd in different organs [23] and that has been used in a previous animal study [9]. Skin Gd concentration was determined using laser ablation-inductively coupled plasma-mass spectrometric imaging (LA-ICP-MSI) as shown in Fig. 1. Laser ablation allows a subsequent spatially resolved elemental analysis via inductively coupled plasma-triple quadrupole mass spectrometry (ICP-TQMS) especially for metals in various tissues [23, 24]. A laser spot size of 25 µm and a corresponding stage speed of 100 µm/s were selected for high-throughput ablation. The formed aerosol is atomized in the plasma, and analyzed in the mass spectrometer, partly after reaction to the detected species (e. g., 158Gd16O+) in the triple quadrupole mass analyzer. Using an appropriate software package, the transient signal of the ICP-MS is used to reconstruct the spatial distribution of the analytes within the biopsy samples (Fig. 1).

Fig. 1figure 1

Microscopic images (A, C, E) of skin biopsy samples and the LA-ICP-TQMS- based detection of Gd (B, D, F) with a likelihood of prior GBCA injection being unlikely (A, B), possible (C, D), and likely (E, F). NER normalized event rate

To evaluate samples with overall low expected Gd concentrations, as in the case of human skin biopsies, a script-based semi-quantitative approach was developed, which introduces the Normalized Event Rate (NER) as an indicator for the real Gd concentration. Utilizing this value, all patients were classified, reflecting their likelihood of prior GBCA injection: lower than 3xstandard deviation (SD) of the controls (unlikely), greater than 3xSD and lower than 10xSD of the controls (possible), and greater than 10xSD of the controls (likely). For further information regarding the calculation of the NER, please refer to the supplemental material (Supplemental Material 1). To analyze possible group differences or associations, we used the frequency of patients within the classes (unlikely, possibly, and likely) as well as the individual NER as a more quantitative estimate of Gd in the tissue.

Phenotyping of painQuantitative sensory testing (QST)

Quantitative sensory testing (QST) was performed according to the protocol of the German Research Network on Neuropathic Pain (DFNS) in 18 (55%) patients with iSFN [25, 26]. ISFN patients with or without Gd were compared to the normative data set of the German network on neuropathic pain (DFNS) and with each other [27]. A total of 11 parameters were used in the analyses: the thermal detection thresholds for the perception of cool (CDT) and warm (WDT), the thermal pain thresholds (cold pain threshold [CPT]; heat pain threshold [HPT]), the mechanical detection thresholds (MDT), the mechanical pain thresholds (MPT), a stimulus–response function for mechanical pain sensitivity (MPS), pain in response to light touch (dynamic mechanical allodynia [DMA]), the vibration detection threshold (VDT), the wind-up ratio (WUR) to assess pain summation to repetitive pinprick stimuli and the pressure pain threshold (PPT) at the thenar eminence.

QST data were z-transformed into a standard normal distribution (zero mean, unit variance) for each single parameter to allow a comparison of QST parameters independent of their physical units using the following expression (except DMA): Z = (value patient – mean controls)/SDcontrols. Z-scores below zero indicate a loss of function; z-scores above zero indicate a gain of function. Thus, elevations of thresholds (CDT, WDT, HPT, CPT, PPT, MPT, MDT, and VDT) result in negative z-scores, whereas increased ratings (MPS and WUR) result in positive z-scores.

Pain questionnaires

The current, maximum, and mean pain intensity of the last 4 weeks was obtained on a numeric rating scale in every SFN patients (anchors: 0 = no pain; 10 = worst pain imaginable). Pain quality and distribution was assessed using the German Pain Questionnaire of the German pain society as a section of the international society for the study of pain [28].

Statistical analysis

For QST parameters, comparisons to the normative data were performed using t-tests as recommended [27]. Since only 2 patients reported DMA, no further analysis was calculated for this parameter. However, due to the small sample size bootstrapping (number of samples = 1000) procedure was used for the one sample t test using SPSS version 28.0 for Mac OS X (IBM Corp. Released 2021. IBM SPSS Statistics for Macintosh, Version 28.0. Armonk, NY, USA: IBM Corp). The iSFN patient with (iSFNe; exposed) and without (iSFNne; not exposed) confirmed GBCA exposure were compared by non-parametric Mann–Whitney-U-tests. Statistical evaluation of the LA-ICP-MSI-derived NERs and the z-transformed IENFD values was performed by non-parametric test (Kruskal–Wallis test followed by Dunn’s multiple comparisons test) using GraphPad Prism version 9.4.0 for Mac OS X (GraphPad Software, San Diego, CA, USA). Nominal or ordinal variables were analyzed by frequency tables and Chi2 tests as well as rank correlation analyses using SPSS version 28.0. For the analyses, the significance criterion was set to p = 0.05 and multiple comparisons were adjusted to the number of comparisons (Bonferroni correction).

Data availability

Data are available in the tables.

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