Non-invasive evaluation of nigrostriatal neuropathology in a proteasome inhibitor rodent model of Parkinson's disease

Abstract

Background: Predominantly, magnetic resonance imaging (MRI) studies in animal models of Parkinson's disease (PD) have focused on alterations in T2 water 1H relaxation or 1H MR spectroscopy (MRS), whilst potential morphological changes and their relationship to histological or behavioural outcomes have not been appropriately addressed. Therefore, in this study we have utilised MRI to scan in vivo brains from rodents bearing a nigrostriatal lesion induced by intranigral injection of the proteasome inhibitor lactacystin.

Results: Lactacystin induced parkinsonian-like behaviour, characterised by impaired contralateral forelimb grip strength and increased contralateral circling in response to apomorphine. T2-weighted MRI, 3-weeks post-lesion, revealed significant morphological changes in PD-relevant brain areas, including the striatum and ventral midbrain in addition to a decrease in T2 water 1H relaxation in the substantia nigra (SN), but not the striatum. Post-mortem histological analyses revealed extensive dopaminergic neuronal degeneration and alpha-synuclein aggregation in the SN. However, extensive neuronal loss could also be observed in extra-nigral areas, suggesting non-specific toxicity of lactacystin. Iron accumulation could also be observed throughout the midbrain reflecting changes in T2. Importantly, morphological, but not T2 relaxivity changes, were significantly associated with both behavioural and histological outcomes in this model.

Conclusions: A pattern of morphological changes in lactacystin-lesioned animals has been identified, as well as alterations in nigral T2 relaxivity. The significant relationship of morphological changes with behavioural and histological outcomes in this model raises the possibility that these may be useful non-invasive surrogate markers of nigrostriatal degeneration in vivo.

Figure 1

Assessment of neurological and behavioural deficits. (A) Neurological scoring (B) Percentage of lactacystin-lesioned animals that are impaired in each of the neurological score tests at days 3, 7 and 21. (C) No significant reduction in forelimb grip strength was observed in saline-injected controls. By contrast in lactacystin-lesioned animals (D) forelimb grip strength was significantly reduced in the contralateral forelimb at 1 and 3 weeks post-lesion. Data shown are mean forelimb grip force (g) ± SEM *p < 0.01 saline vs. lactacystin. (C) Lactacystin-lesioned animals displayed significantly increased rotational asymmetry in response to apomorphine challenge (0.1 mg/kg s.c.), Data shown are mean area under curve (AUC) ± SEM ***p < 0.001 saline vs. lactacystin.

Figure 2

(A) Representative T2W MR images from saline-injected control subject with sample ROIs utilised for quantitative volumetric analysis of individual brain regions. (B-G) Bar graphs of regional brain volumetric data measured on in vivo T2W anatomical MRI scans acquired from saline and lactacystin-injected animals 3 weeks post-lesion. (B) Whole brain volume, (C) corpus striatum, (D) ventral midbrain, (E) lateral ventricles, (F) cerebellum and (G) hippocampal formation. Data are expressed as mean volume mm³ ± s.e.m. *p < 0.05, **p < 0.01; ipsilateral vs. contralateral hemisphere; *p < 0.01; ipsilateral hemisphere of lactacystin vs. saline-injected; §p < 0.05 contralateral hemisphere of saline vs. lactacystin-injected.

Figure 3

Representative T2W MR images through the striatum (panel A) and midbrain containing the substantia nigra (SN; panel B) to illustrate regional brain atrophy in saline and lactacystin-injected animals. Clear hypertrophy of both the ipsilateral (solid arrows, A) and contralateral (dashed arrows, A) LV can be observed in lactacystin-lesioned animals, compared to saline controls. In the midbrain (panel B) the sub-cortical area containing the SN in the ipsilateral hemisphere appears clearly deformed compared to the intact contralateral hemisphere (solid arrows). Note also the increased CSF signal, suggestive of a volume change in this region. In addition, areas of T₂ hypointensity are observed in the SN, but also in extra-nigral nuclei (dashed arrows).

Figure 4

Bar graphs of T₂ relaxivity measured from parametric T₂ maps generated from T2W MRI scans acquired 3 weeks post-lesion from saline and lactacystin-injected animals. (A) No significant difference in T₂ was observed in the striatum between saline and lactacystin-injected animals. By contrast, moderate, but significant reductions in T₂ were observed in the ventral midbrain (C) and substantia nigra (E) of lactacystin-injected animals compared to saline controls. Example ROIs used to generate quantitative T₂ data are shown for striatum in (B), ventral midbrain (D) and substantia nigra (F), respectively. Data shown are the ratio of T₂ between the contralateral and ipsilateral hemispheres ± SEM for saline and lactacystin-injected animals, respectively. *p < 0.05; saline vs. lactacystin.

Figure 5

Histological assessment of nigrostriatal integrity 3 weeks post-lesion. Quantification by unbiased stereology revealed intranigral injection of lactacystin resulted in a marked decrease in (A) the number of TH+ cell bodies in the ipsilateral SNc, mirrored by a clear reduction in (B) NeuN+ cells. ***p < 0.001; contralateral vs. ipsilateral hemisphere, #p < 0.01; ipsilateral hemisphere of lactacystin vs. saline controls. (C, D) Representative photomicrographs of TH+ staining in saline and lactacystin-lesioned animals. Note the extensive loss of TH+ and NeuN+ cells in the ipsilateral SNc, but also in the VTA, SNr and extranigral nuclei in proximity to the injection tract of lactacystin-injected animals (arrows) (Magnification (C) × 1.5, scale bars 500 μm; (D) ×4, scale bars 200 μm). Concomitant with nigral cell body degeneration, a substantial dennervation of TH+ fibres was observed in the ipsilateral striatum of lactacystin-lesioned animals quantified by optical densitometry (E) and illustrated by a representative photomicrographs in (F) (magnification ×1.5, scale bar 500 μm; insets ×40, scale bar 20 μm). ***p < 0.001; contralateral vs. ipsilateral hemisphere, #p < 0.01; ipsilateral hemisphere of lactacystin vs. saline controls.

Figure 6

Lactacystin lesioning does not result in loss of striatal medium spiny neurons. (A) Whole brain photomicrogaphs of NeuN and DARPP-32+ cells in the striatum of saline and lactacystin lesioned animals (×1.5 magnification, scale bar = 500 μm). (B) Higher magnification of NeuN+ and DARPP-32+ cells in the striatum of saline and lactacystin injected animals, respectively. (×40 magnification, scale bar = 20 μm). Note the enlarged ventricles in lactacystin-injected animals (asterisks in A).

Figure 7

(A-D) α-synuclein immunohistochemistry reveals the presence of soluble aggregates of α-synuclein in the substantia nigra of lactacystin-lesioned animals compared to saline controls (panels A, B: ×4 magnification, scale bars = 200 μm; panels C, D: ×40 magnification, scale bars = 20 μm). Note the diffuse pattern of staining in control animals compared to the punctate labelling observed in lactacystin-lesioned animals, indicative of aggregation. (E-F) Pre-treatment with proteinase K (10 μg) reveals the presence of insoluble perinuclear α-synuclein aggregates in the SNc of lactacystin-injected animals, but not saline controls (×60 magnification, scale bars = 10 μm).

Figure 8

(A) Prussian blue and cresyl violet histology reveals no apparent iron accumulation in the striatum of both saline and lactacystin-lesioned animals (arrowheads in A) (×2.5 magnification, scale bars = 300 μm; insets × 40 magnification, scale bars = 20 μm). (B) By contrast, extensive iron deposits are observed in the SNc of lactacystin lesioned animals but not saline controls (arrowheads in B); (×2.5 magnification, scale bars = 300 μm; insets ×40 magnification, scale bars = 20 μm). Note that iron deposition is not confined to the SN but is also present in extra-nigral regions (C) Densitometry measurements confirmed the increased in iron deposition in the SN of lactacystin-lesioned animals compared to saline controls. Data shown are mean optical density values (arbitrary units) ± SEM. **p < 0.01; saline vs. lactacystin. (D) Iron deposits were also observed in proximity to the needle-tract of lactacystin-lesioned animals, but not saline controls (arrows in D) (×2.5 magnification, scale bar = 300 μm). Interestingly, iron accumulation appears to overlap with areas of neuronal loss in the midbrain of lactacystin-lesioned animals as shown in Figure 5D.