Deposition of Gadolinium in the Central and Peripheral Nervous Systems and Its Effects on Sensory, Cognitive, and Athletic Implications after Multiple Injections of Gadolinium-Based Contrast Agents in Rats

Animals and Study Design

Forty-eight male Wistar rats (220–240 g, 6 weeks of age) used in the experiments were purchased from the Xiamen University Laboratory Animal Center. The animals were housed in standard laboratory conditions with free access to water and food under a 12-hour light-dark cycle. Before the formal experiment, the rats were underwent for 7 days for acclimatization. All experimental studies were approved by the Animal Ethics Committee of Xiamen University.

The Wistar rats were randomly divided into 4 experimental groups (12 rats in each group): the gadodiamide group (linear and nonionic GBCAs, Omniscan, 500 mmol Gd/L; GE Healthcare), the gadopentetate dimeglumine group (linear and ionic GBCAs, Magnevist, 500 mmol Gd/L; Bayer HealthCare Pharmaceuticals), the gadoterate meglumine group (macrocyclic and ionic GBCAs, Dotarem, 500 mmol Gd/L; Guerbet), and a control group (0.9% saline solution). GBCAs were injected via the tail vein for 4 consecutive days per week for 4 weeks at a daily dose of 0.6 mmol/kg.⁹ This dose was equivalent to the normal human dose of 0.1 mmol/kg recommended by the FDA. The control group received the same equivalent of 0.9% saline solution. A flow chart of MR scanning and the administration of GBCAs is presented in Fig 1.

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FIG 1.

Flow chart of experimental design. The experimental timeline of GBCA injections, MR scanning (days 0, 7, 14, 21, 28, and 42), inductively coupled plasma mass-spectroscopy (day 28), TEM (day 42), heat and mechanical hyperalgesia tests (days 0, 28, and 42), the accelerated rotarod test and the Morris water maze test (days 0, 28, and 42), immunohistochemical test (days 28 and 42), and euthanasia time point. In total, 48 rats were used in the experiment. The number of rats used in each experiment is indicated in the diagram. M-maze indicates the Morris water maze test; inj., injection.

MR Imaging and Analysis

The rats were anesthetized (2.0% isoflurane) and placed on a mechanical ventilator. Mean body temperature was maintained at 37.0°C [SD, 0.5°C] using a heated circulating water pad. The rats were placed in the prone position in a stereotactic stent for MRI. The MRI experiments were performed on a horizontal-bore 9.4T scanner operating on an AVANCE platform (Bruker 9.4T Biospec). The ventilation unit continuously ventilates isoflurane gas while scanning. T1-weighted MRI with a fast spin-echo sequence was performed on the brains of the rats (field of view = 50 × 50 mm, bandwidth = 348.77 kHz, matrix size = 256 × 256, scan slices = 15, slice thickness = 1.0 mm, TE = 8.5 ms, TR = 1366.9 ms, and number of excitations = 15).^10,11

After MRI scanning the original images were sent to the 9.4T Micro MRI experimental workstation (Bruker 9.4T Biospec) for image data analysis. All images were analyzed in blinded and randomized fashions. The ROIs were located in the bilateral DCN and cerebellum of the rat brain. Quantitative evaluations of the DCN T1 signal intensity were performed by positioning the ROIs over the bilateral DCN and cerebellum zones according to the anatomy of the rat brain. The DCN T1 signal intensity was quantitatively evaluated. The signal intensity ratio was calculated as follows:^12,13

Inductively Coupled Plasma Mass-Spectroscopy Analysis for Gd Detection and Quantification

Rats were euthanized with excessive sodium pentobarbital anesthesia. Subsequently, the heart was exposed and rapidly perfused with 0.9% saline solution through the left atrium to remove excess blood from the brain. After the liver completely turned white, the sample tissue was quickly removed. Cerebellar, whole spinal cord, and peripheral nerve (sciatic nerve) samples were isolated according to the anatomic atlas of rats and extracted on ice.¹³ The samples were weighed (about 200 mg) and sealed in quartz tubes, immersed in 2.0 mL of concentrated nitric acid, and subjected to digestion for 90 minutes. Then, each sample was transferred to a polypropylene tube and diluted to 10 mL with ultrapurified water. Gd standard solution and internal standard solution were prepared as quality controls. Subsequently, the accumulation of 158 in each sample was measured using inductively coupled plasma mass-spectroscopy (ICP-MS) (7700x; Agilent Technologies; internal standard indium 115). The concentration of Gd was monitored using a standard curve of inorganic Gd (0.1–50 μg/L) and expressed as nanomoles of Gd per gram of wet tissue weight.

Transmission Electron Microscopy Analysis for the Observation of Gd Deposition

Rats were euthanized with excessive sodium pentobarbital anesthesia. The cerebellum, whole spinal cord, and sciatic nerve were separated as soon as possible on ice according to the rat anatomy atlas and washed with cold saline. Then, the spinal cord tissue was quickly fixed with 2.5% glutaraldehyde, and 1-mm³ samples were collected. The samples were rinsed in 0.1 mol/L Na-cacodylate buffer (pH = 7.4) 3 times, fixed in 1% osmic acid for 2 hours, and then washed with saline 3 times. The samples were then dehydrated with graded ethanol and acetone series, immersed in Epon812 epoxy resin (Beyotime, Shanghai, China) for 12 hours, dried, and polymerized in an oven at 70°C for 24 hours. Next, ultrathin sections (0.1 μm) were cut and stained with 2% lead citrate glued to a copper grid. The ultrastructure was observed by transmission electron microscopy (TEM). Elemental composition analysis used energy-dispersive x-ray, and the distribution of concentrated GBCA particles was observed.

Heat Hyperalgesia and Mechanical Hyperalgesia Test

The test was conducted by radiant heat stimulation applied to the plantar surface of the hind paw to produce a pain response at a set temperature (intensity of 40 infrared units). When the rat received the stimulation to withdrawal the paw, it was regarded as a pain response, and the withdrawal latency was the time from stimulus onset to paw withdrawal. About 30 minutes before the test, the rats were individually placed in clear plastic cages for accommodation. Heat stimuli were applied 3 times with a 10-minute rest between trials. The averages of 3 times withdrawal latency were recorded. The experiment was performed before the injection of GBCAs and at days 28 and 42 after the first injection of GBCAs.

A von Frey filament (Ugo Basile, Milan, Italy) with a bending force of 15 g was applied to the plantar surface of the rat hind paw, and the frequency of paw withdrawal was recorded. At the beginning of the test, the plantar surface was poked successively 5 times at 5-minute intervals, and the paw withdrawal frequency was reported. The mean of the 5 tests was calculated. The experiment was performed before the injection of GBCAs and on days 28 and 42 after the injection of GBCAs.

Accelerated Rotarod Test and Morris Water Maze Test

The assessment of the Morris water maze task was slightly modified as previously described.¹⁴ The rats were screened before the experiment to identify those that could not swim. The tests were conducted before the injection of GBCAs and at days 28 and 42 after the injection of GBCAs twice daily. In each test, all animals were tasked with finding a hidden, transparent plastic platform (10 cm in diameter) placed 50 cm from the water maze wall (150 cm in diameter and 60 cm in depth) and 1 cm below the water. The test was done blindly, and all animals were randomized in each group. The rats faced the pool wall before being released, and the time it took to reach the hidden platform was recorded. The rats were allowed to rest on the platform for 30 seconds during the test.

The abilities of motor skills and coordination in rats were measured with an accelerated rotarod apparatus. The speed was accelerated from 0 to 40 rpm for >4 minutes.¹⁵ The rats received once-daily training sessions of 3 trials separated by 30-minute intervals for 3 consecutive days before the injections of GBCAs. The longest time each rat stayed on the rod was recorded as the baseline. At days 28 and 42 after the injections of GBCAs, rats were tested 3 times on the rotarod, and the best performance of each rat was recorded on that day.

Histologic Observations

Rats were euthanized with an overdose of sodium pentobarbital anesthesia. Subsequently, the spinal cords were isolated and extracted on ice quickly and then fixed in 4% buffered paraformaldehyde for 48 hours. The samples were then dehydrated, embedded in paraffin, cut into 5-μm sections, and stained with H&E, Nissl, neuron-specific enolase (NSE, 1:1000, 24330; CST), and glial fibrillary acidic protein (GFAP, 1:1000, ab7260; Abcam) stains. GFAP is a type III intermediate filamentous protein mainly distributed in astrocyte glial cells of the nervous system and is a marker of astrocyte activation. In the case of neuronal injury, GFAP expression is elevated, suggesting astrocyte gliosis.¹⁶ In the nervous system, NSE is a soluble cytoplasmic protein that is continuously expressed by mature neurons and neuron-derived cells. It is a brain tissue–specific glycolytic enzyme that plays an important role in the process of cellular energy metabolism. Elevated NSE expression can be detected in nervous system injury.¹⁷ Immunohistochemical analysis was performed microscopically by Xiang Yao with >10 years of experience (M2700 P; Leica). Histologic evaluation of the spinal cord was performed, including the observation of neuronal cell degeneration and obvious necrosis and the expression of NSE and GFAP. The evaluators were blinded to the treatment groups. ImageJ software (National Institutes of Health) was used to analyze the results.

Statistical Analysis

All statistical analyses were performed by using SPSS 17.0 (IBM) or GraphPad Prism 8 software (GraphPad Software), and the data are expressed as mean (SD). For the analysis of immunohistochemistry and Gd deposition, 1-way ANOVA was used, followed by post hoc analysis with the Tukey test. For the sensory tests, a mixed-design ANOVA was performed. One-way ANOVA was used to detect heat hyperalgesia and mechanical hyperalgesia experiments, and the Dunnett test was used for post hoc analysis. P < .05 was considered a significant difference.