Transgenic rat model of Huntington's disease: a histopathological study and correlations with neurodegenerative process in the brain of HD patients

Abstract

Rats transgenic for Huntington's disease (tgHD51 CAG rats), surviving up to two years, represent an animal model of HD similar to the late-onset form of human disease. This enables us to follow histopathological changes in course of neurodegenerative process (NDP) within the striatum and compare them with postmortem samples of human HD brains. A basic difference between HD pathology in human and tgHD51 rats is in the rate of NDP progression that originates primarily from slow neuronal degeneration consequently resulting in lesser extent of concomitant reactive gliosis in the brain of tgHD51 rats. Although larger amount of striatal neurons displays only gradual decrease in their size, their number is significantly reduced in the oldest tgHD51 rats. Our quantitative analysis proved that the end of the first year represents the turn in the development of morphological changes related to the progression of NDP in tgHD51 rats. Our data also support the view that all types of CNS glial cells play an important, irreplaceable role in NDP. To the best of our knowledge, our findings are the first to document that tgHD51 CAG rats can be used as a valid animal model for detailed histopathological studies related to HD in human.

Figure 1

(a) Lateral brain ventricles are quite narrow in young (3-month-old) control wt rats, unlike (b) notably enlarged ventricles in 18-month-old tgHD51 rats, owing to the progression of striatal atrophy which confirms the development of NDP. Haematoxylin and eosin. Direct magnification 1.5x.

Figure 2

Significant decrease in the size (diameter) of the striatal neurons in a course of the development of NDP is marked by accompanied changes in the diameter of their nuclei, due to the maintenance of the nucleo-cytoplasmic rate. Detection of the β-III-tubulin⁺ neurofilaments, which fill in the entire cytoplasm of neuronal body and processes, enables to document the shrinkage not only of neuronal nuclei but also of the bodies of neurons, due to either physiological ageing process (a–c) or that caused by the progression of neuronal degeneration in tgHD rats (d–f). The number of GFAP⁺ astrocyte grows up only slightly in course of ageing (a–c), but significant increase (astrogliosis) is evident in oldest tgHD rats (f). (a) wt—2-month-old, (b) wt—12-month-old, (c) wt—22-month-old, (d) tgHD—6-month-old, (e) tgHD—12-month-old, (f) tgHD—24-month-old rats. Anti-β-III-tubulin (red) + anti-GFAP (green) + DAPI (blue). Bar 20 μm.

Figure 3

Significant difference in the number and size of neurons/neuronal nuclei (NeuN⁺) is evident if we compare (a) 2-month-old wt rats and (b) 18-month-old tgHD rats; (b) concomitant reactive astrogliosis is already developed in 18-month-old tgHD rats; it is also apparent that the degeneration of neurons in tgHD rats is typically selective (alike in human HD brain). Anti-NeuN (red) + anti-GFAP (green) + DAPI (blue). Bar 50 μm.

Figure 4

In postmortem samples of human HD brain, (b, c) gradual progression of chronic striatal NDP marked by massive neuronal degeneration and severe concomitant astrogliosis is evident in comparison with (a) intact control brain. (a) Control (♂/56), (b) HD duration 8 years, grade 3 (♂/38), and (c) HD duration 20 years, grade 4 (♀/52). Anti-NeuN (red) + anti-GFAP (green) + DAPI (blue). Bar 20 μm.

Figure 5

(a) Progress Chart: the progression in decrease of the median diameter of neuronal nuclei with age of rats in wt groups (black line) and tgHD51 rats (grey line). (b) Box Plot: statistical characteristic of the groups of rats. The multiple comparison of the median diameter of neuronal nuclei of the groups of rats: “young_wt” and “young_tgHD” are groups of rats 3 and 6 months (0–6 months) old; “old_wt” and “old_tgHD” are groups of rats 12, 18, and 24 months old (>6 months).

Figure 6

(a) In young control animals (2 months old), synapses within the neuropil are very numerous, fine and of uniform size; (b) the most conspicuous alteration in old (here 18 months) tgHD rats is variable size and enlargement (coarsening) of most of synapses; however, also their decreased number participating in loosening of neuropil is evident. Anti-synaptophysin counterstained with 0.1% methyl green. Bar 10 μm.

Figure 7

(a) Synapses in intact (control, ♂/56) human brain are (like in rats) uniform and densely accumulated within the neuropil; (b) they also become coarser and of variable size with the progression of NDP in HD patients (here ♀/52, grade 4, duration 20 years); continuous decrease in their number significantly participates in rarefaction of the neuropil, most prominent in terminal stage of NDP. Anti-synaptophysin counterstained with 0.1% methyl green. Bar 10 μm.

Figure 8

(a) Only fine polyQ deposits are spread in the nuclei of striatal neurons in control 6-month-old rats unlike (b) a higher density of polyQ deposits in age-matched tgHD rats; (c) significantly increased density of polyQ inclusions, in both neurons and some glial cells (yellow arrows), is already in 12-month-old tgHD rats and (d) particularly in aged (here 18-month-old) tgHD rats; increased density of polyQ expression is probably also influenced by the shrinkage of nuclei during the degeneration of neurons; the progression of rarefaction of the neuropil is also apparent. (e) In the cortex of 2-month-old wt rats, perinuclear polyQ positivity is only in some neurons, unlike increased concentration of polyQ (mhtt) deposits particularly within the cytoplasm, primarily of pyramidal neurons, in (f) 12-month- and (g) 18-month-old tgHD rats; in aged tgHD rats, the significant number of degenerated (hyperchromic) neurons is present (blue arrows); only few polyQ⁺ and subsequently degenerated glial cells (yellow arrows) and particularly loosening of the neuropil are also characteristic for the progression of NDP in the cortex of tgHD51 rats. Anti-polyQ counterstained with 0.1% methyl green. Bar 20 μm.

Figure 9

(a) In young (2-month-old) wt rats S100β ⁺ astrocytes are predominantly GFAP-negative; with the progression of NDP in (b) 18-month-old tgHD rats and (c) 24-month-old tgHD rats the main GFAP⁺ processes become coarser, albeit less branched, and the most distinct are their vascular end-feet (thickening of the perivascular membrane in form of “rings”); the number of not only S100β ⁺/GFAP⁻ but also S100β ⁺/GFAP⁺ astrocytes increases primarily around the vessels; the coexpression is seen mainly within the thick astrocytic processes terminating by the end-feet on the vessel wall. Anti-GFAP (red) + anti-S100β (green) + DAPI (blue). Bar 20 μm.

Figure 10

In HD human brain, significant “reactive” astrogliosis develops with the progression of NDP (b, c) in comparison with control brain (a); also the fine GFAP⁺ processes become numerous and of characteristic arborization; specific terminal swellings are in most of the densely GFAP⁺ processes; although the amount of S100β ⁺/GFAP⁻ is not significantly influenced by NDP, the regressive changes (shrinkage) affect also S100β ⁺ cells. (a) Control (♂/56), (b) HD duration 8 years, grade 3 (♂/38), and (c) HD duration 20 years, grade 4 (♀/52). Anti-GFAP (red) + anti-S100β (green) + DAPI (blue). Bar 20 μm.

Figure 11

NG2 glia forms a dense 3D network, particularly prominent on thick (30 μm) slices of the rat brain; there is no significant alteration in density and arrangement of this network if we compare control wt rats with age-matched tgHD rats. (a, b) 18-month-old wt rat; (c) 18-month-old tgHD rat. Anti-NG2. Bar (a) 50 μm and (b, c) 20 μm.

Figure 12

(a) Microglia (green) in the striatum of young (6-month-old) wt rats were mostly of small size, that is, nonactivated, although some neurons are here also scavenged (physiological degeneration); (b) the number of microglia increases quite slowly with the progression of NDP in tgHD rats; however (c) significant increase is only in the oldest animals (from 18 months of age). The double-staining with MAP2 antibody enables to document different stages of the degeneration of striatal neurons (red) up to the removal of their remnants; moreover, the accumulation of debris (red) in the cytoplasm of microglia (green); (b, c) 1—inactive microglia, 2—scavenged neuron (early stage of phagocytic process), and 3—remnants of the ingested material (phagosomes) in the cytoplasm of microglia. Additionally, significant gradual reduction in the number of dendrites (i.e., rarefaction of the neuropil) with the progression of NDP is evident in aged tgHD rats. (b) tgHD—12-month-old rat; (c) tgHD—18-month-old rat. Anti- MAP2 (red) + anti- Iba1 (green) + DAPI (blue). Bar 20 μm.

Figure 13

(a) In control human brain (♂/33), microglia (red) are mostly inactive/small (here in CN), but (b) their number and size significantly increase in advanced stage of NDP in HD brain (CN) (♀/52, HD duration 20 years, grade 4); (c) degenerated neurons within the CN are scavenged by microglia labelled with densely red stained lysosomes inside their cytoplasm (♂/38, HD duration 8 years, grade 3); (d) in advanced stage of NDP (♀/52, HD duration 20 years, grade 4), almost all neurons in CN already degenerated and they were replaced by reactive astrocytes; S100β ⁺ astrocytes also form a typical network; however, the neuropil is only sparse; microglia are numerous, particularly related to vessels (yellow arrows). (a, b, and c) Anti-Iba1 (red) + DAPI (blue); (d) anti-Iba1 (red) + anti-S100β (green) + DAPI (blue). Bar: (a, b) 50 μm; (c, d) 20 μm.