Long-term effects of linear versus macrocyclic GBCAs on gene expression in the central nervous system of mice

Abstract

Background: We examined chronic gadolinium retention impact on gene expression in the mouse central nervous system (CNS) after injection of linear or macrocyclic gadolinium-based contrast agents (GBCAs).

Methods: From 05/2022 to 07/2023, 36 female mice underwent weekly intraperitoneal injections of gadodiamide (2.5 mmol/kg, linear), gadobutrol (2.5 mmol/kg, macrocyclic), or saline. Mice were sacrificed on day 29 or 391 after a 1-year washout. Assessments included magnetic resonance imaging (MRI), mechanical hyperalgesia tests, and inductively coupled plasma mass spectrometry to measure gadolinium levels. Ribonucleic acid (RNA) sequencing and bioinformatic analyses identified differentially expressed genes (DEGs), with validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot (WB).

Results: Post-gadodiamide, MRI showed increased signal intensity in the deep cerebellar nuclei (pre, 0.997 ± 0.006 versus post, 1.086 ± 0.013, p < 0.001). Mechanical hyperalgesia tests indicated transient sensory changes. After 1-year, gadolinium retention was noted in the brain (5.92 ± 0.32 nmol/kg) and spinal cord (1.23 ± 0.66 nmol/kg) with gadodiamide, compared to saline controls (0.06 ± 0.02 nmol/kg in brains and 0.28 ± 0.06 nmol/kg in spinal cords). RNA sequencing identified 17 shared DEGs between brain and spinal cord in the gadodiamide group on day 391, with altered Hmgb2 and Sgk1 expression confirmed by qRT-PCR and WB. Reactome pathway analysis showed enrichment in neuroinflammation pathways. No DEGs were detected in brains on day 29.

Conclusion: Chronic gadolinium deposition from repeated linear GBCA but not macrocyclic administration causes significant gene expression alterations in the mouse CNS, particularly affecting neuroinflammation pathways.

Relevance statement: This study examined the long-term impact of chronic gadolinium retention on gene expression in the mouse CNS, uncovering significant changes associated with neuroinflammation pathways after repeated administration of linear GBCA, but not with macrocyclic GBCA. These findings highlight the importance of further research on the long-term safety of linear GBCA in medical imaging.

Key points: Chronic gadolinium retention alters gene expression in the mouse central nervous system. Significant neuroinflammatory pathway changes were observed after linear gadodiamide exposure. MRI showed increased signal intensity in deep cerebellar nuclei after gadodiamide injection.

Keywords: Animals; Central nervous system; Contrast media; Gadolinium; Gene expression.

Fig. 1

Global scheme of group assignments for gadolinium-based contrast agent injections and the following analysis

Fig. 2

a Representative T1-weighted images at days 0, 29, and 391. Arrows indicate areas of increased signal intensity. b Quantitative signal intensity ratio of the deep cerebellar nuclei (DCN) and cerebellar hemisphere (CH) at days 0, 29, and 391

Fig. 3

Time course of mechanical hyperalgesia (number of withdrawals per trial) in the right and left hind paw of mice treated with gadodiamide (2.5 mmol/kg), gadodiamide, gadobutrol (2.5 mmol/kg), and saline. Data are expressed as mean ± standard error of the mean (six rats per group). **** indicates p < 0.001

Fig. 4

Gadodiamide versus saline: Volcano plots of differential gene expression for the brain on day 29 (a), the brain on day 391 (b), and the spinal cord on day 391 (c). Gadobutrol versus saline: Volcano plots for the brain on day 29 (d), the brain on day 391 (e), and the spinal cord on day 391 (f). Red dots indicate differentially expressed genes with adjusted p < 0.05 and |log2FC | > 1

Fig. 5

Relative mRNA expression of Hmgb2 and Sgk1. Brain at day 29 (a, b), brain at day 391 (c, d), Spinal cord at day 391 (e, f) after injection with gadodiamide, gadobutrol, and saline. ns indicates p > 0.05, *** indicates p < 0.001

Fig. 6

Relative protein levels of HMBG2 and SGK1. Brain at day 29 (a, b), brain at day 391 (c, d), Spinal cord at day 391 (e, f) after injection with gadodiamide, gadobutrol, and saline. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01