Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a gradual loss of dopaminergic neurons in the substantia nigra pars compacta¹. We investigated the utility of magnetic resonance imaging (MRI) in detecting macroscopic structural brain changes in the unilaterally-lesioned 6-hydroxydopamine (6-OHDA) model.

Methods

After the approval of Institute Animal Ethics Committee, adult male Wistar rats (n = 12, weighing 250–300 g) were divided into PD and Sham groups (n=6 in each). Two injections of 2 µL of 6-OHDA solution (7.5 µg/µL 6-OHDA dissolved in 0.2% ascorbate saline) were given in the right hemispheric striatum in the coordinates with reference to the bregma². The sham-lesioned group went through the same procedure with the injection of saline (instead of 6-OHDA). Necessary postoperative care was provided and all procedures were performed as per the guidelines of the Committee for Control and Supervision of Experiments on Animals (CCSEA).
The extent of the 6-OHDA lesioning was confirmed by the apomorphine-induced rotational test. Rats received a subcutaneous injection of apomorphine (0.1 mg/kg in 0.9% saline, E-Biomed GmbH, Heidelberg, Germany). After 5 min, the rat was placed in a hemispherical bowl for 30 minutes and monitored with the help of the tracking software (XT software, EthoVision, The Netherlands). The number of ipsiversive and contraversive full-body turns was recorded automatically over a period of 30 minutes.
High-resolution T1- weighted 3D gradient-echo MRI sequence (RF-spoiled fast low-angle shot (FLASH), repetition time (TR)/ echo time (TE) = 317.89/4.5ms, flip angle = 30°; voxel size = 0.0684 x 0.0684x0.8mm³; number of averages = 3.
Each image was reconstructed using Bruker’s Paravision software, exported in DICOM format, and converted to NIfTI format, manually rotated to match the standard stereotaxic space in rat³, and resized by a factor of 10 to account for the brain size difference between rodents and humans; further aligned to the stereotaxic space by registering each image to the template image using “Coregister” tool in SPM12 and was resampled into 0.125-mm isotropic voxels and then segmented into probability maps of gray matter (GM), white matter (WM), and cerebrospinal fluid with rat tissue priors using SPM8 (Figure 1). The resulting GM and WM volumetric maps were then used in the second level analysis (paired t-test for intra-group comparisons and two-sample t-test for inter-group comparisons) to estimate the differences in atrophy between the groups.

Results

A contralateral rotational response to apomorphine was exhibited by the 6-OHDA PD model at 3rd and 7th week post-surgery (confirming the lesion) compared to sham control (Figure 2).
VBM revealed widespread bilateral changes in GM volume on a topographic scale in the brains of 6-OHDA rats, compared to sham-operated model. A reductions of GM volume were observed in fimbria (ipsi), hippocampal formation (ipsi) and accumbens nucleus (contra) in 3rd week PD and cerebellum (ipsi and contra both) in 7th week PD as compared to sham. Additionally, GMV revealed a decreasing trend in the retrosplenial granular cortex (c-region) and cerebellum in contralateral hemisphere in the 7th week PD model as compared to 3rd week (p<0.001unc).

Discussion

Behavioural results revealed striatal dopaminergic cell loss and denervation in the striatum. GMV was decreased in cortical and subcortical regions of the ipsilateral (lesioned) and contralateral (unlesioned) hemisphere, including the site of the primary lesion following 6-OHDA lesioning. The reductions observed are similar to that reported in previous studies^4,5 in the third week post-lesion, while the cerebellum exhibited changes in the seventh week post-lesion. This temporal dimension provides a deeper understanding of the progression of structural alterations in response to dopaminergic cell loss.

Conclusion

Unilateral nigrostriatal 6-OHDA lesioning leads to widespread gray matter volume changes, which extend beyond the nigrostriatal system and resemble advanced Parkinsonism.

Acknowledgements

We acknowledge the funding from Department of Health Research (DHR), Govt. of India, Women Scientist Scheme: R.12013/06/2023-HR (for SK) and Department of Health Research (DHR), Govt. of India, Grant No.R.11013/68/2021-GIA/HR