Age and Environment Influences on Mouse Prion Disease Progression: Behavioral Changes and Morphometry and Stereology of Hippocampal Astrocytes

Abstract

Because enriched environment (EE) and exercise increase and aging decreases immune response, we hypothesized that environmental enrichment and aging will, respectively, delay and increase prion disease progression. Mice dorsal striatum received bilateral stereotaxic intracerebral injections of normal or ME7 prion infected mouse brain homogenates. After behavior analysis, animals were euthanized and their brains processed for astrocyte GFAP immunolabeling. Our analysis related to the environmental influence are limited to young adult mice, whereas age influence refers to aged mice raised on standard cages. Burrowing activity began to reduce in ME7-SE two weeks before ME7-EE, while no changes were apparent in ME7 aged mice (ME7-A). Object placement recognition was impaired in ME7-SE, NBH-A, and ME7-A but normal in all other groups. Object identity recognition was impaired in ME7-A. Cluster analysis revealed two morphological families of astrocytes in NBH-SE animals, three in NBH-A and ME7-A, and four in NBH-EE, ME7-SE, and ME7-EE. As compared with control groups, astrocytes from DG and CA3 prion-diseased animals show significant numerical and morphological differences and environmental enrichment did not reverse these changes but induced different morphological changes in GFAP+ hippocampal astroglia. We suggest that environmental enrichment and aging delayed hippocampal-dependent behavioral and neuropathological signs of disease progression.

Figure 1

Experimental timeline. Young adult (6 months old) and aged (18 months old) female albino Swiss mice were maintained for 5 weeks either in enriched or in standard cages and then subjected to injections of normal or ME7 infected brain homogenates and returned to their original cages. After 17 weeks after injections they were submitted to placement and identity object recognition tests, euthanized, and had their brains processed for selective GFAP, PrPSc, and IBA1 immunolabeling.

Figure 2

Burrowing activity. (a) Amount of burrowed food in the ME7-SE group compared to NBH-SE controls. Note significant reduction at 11 wai in ME7-SE compared with NBH-SE and at 15 wai in ME7-EE compared with NBH-EE. (b) ME7-SE young mice were different from ME7-A mice between 13 and 16 wai, and NBH-A showed a reduction in burrowing activity at 16 wai. (^∗p < 0.05, ^∗∗p < 0.01 versus NBH-SE; ^##p < 0.01 versus NBH-EE; ⁺⁺p < 0.01 versus ME7-A; ^§§p < 0.01 versus NBH-SE; Two-way repeated measures Bonferroni posttests).

Figure 3

Object recognition impairments. At 17 wai, all mice were tested on object recognition tasks to recognize placement and identity of objects. (a) ME7-SE, NBH-A, and ME7-A did not distinguish displaced from stationary objects. All other groups succeeded in this task. (b) In the object identity recognition task, except for ME7-A, all groups recognized the identity of the objects (^∗p < 0.05, ^∗∗p < 0.01; two-tailed t-tests).

Figure 4

Photomicrographs from PrPSc and Iba1 immunolabeled sections from the polymorphic layer of dentate gyrus and striatum to illustrate mouse prion disease associated histological changes. Note PrPSc deposits and morphological activated microglia and vacuolation in ME7-SE. Scale bar: 25 μm.

Figure 5

Photomicrographs from control and prion-diseased mice at 18 wai to illustrate morphological astrocytic changes in the stratum radiatum of CA3 from young and aged adults from standard and enriched environments. Scale bar: 25 μm.

Figure 6

Astrocyte number and cell body volume changes. (a) In the polymorphic layer, ME7-SE mice showed higher number of astrocytes than NBH-SE animals. AS compared with NBH-SE, an increased number of astrocytes is observed in NBH-EE. (b) In the stratum radiatum of CA3, NBH-A and ME7-A mice showed higher number of astrocytes than NBH-SE and ME7-SE animals, respectively. In contrast to the polymorphic layer, stratum radiatum of CA3 of ME7-EE showed a higher number of astrocytes than that of ME7-SE mice. (c) and (d) show prion disease influence on the cell body volumes of astrocytes in the DG polymorphic layer and stratum radiatum of the CA3, respectively. ME7-SE and ME7-EE showed significant soma hypertrophy in astrocytes from DG and CA3 compared with their respective controls. However, ME7-A cell body volume was smaller than ME7-SE in the polymorphic layer and stratum radiatum of CA3 layers. In the stratum radiatum of CA3, the cell body volume of astrocytes from ME7-EE mice was smaller than that of ME7-SE animals (^{∗, #}p < 0.05; ^∗∗p < 0.01; ^###p < 0.001 Bonferroni posttests).

Figure 7

Astrocyte morphometry. (a) ME7-SE and ME7-EE mice showed significantly higher branch volumes compared to NBH-SE and NBH-EE animals, respectively. ME7-EE and ME7-A groups had reduced branch volumes versus ME7-SE mice. (c) Prion disease also increased the surface area of astrocyte branches compared with NBH-SE and ME7-A groups. An increase in branch surface area also was seen in ME7-EE versus NBH-EE mice and in NBH-EE versus NBH-SE animals. (e) ME7-SE astrocytes showed significantly longer branches than NBH-SE and ME7-A astrocytes. (b, d) Total volume and total surface area in ME7-SE mice were found in the 1st, 2nd, 3rd, 4th, 5th, and 6th branch orders whereas, in EE animals, effects on surface area were limited to branches from the 1st to 3rd orders, and volume differences were limited to between the 1st and 4th orders. (f) 1st and 2nd branch orders from ME7-EE were significantly longer than the corresponding branches of ME7-SE. Nonsignificant alterations were detected in aged mice (^∗p < 0.05; ^{∗∗, ##}p < 0.01; ^{∗∗∗, ###}p < 0.001 Bonferroni posttests).

Figure 8

Three-dimensional reconstructions of astrocytes of the DG polymorphic layer at 18 wai (a) with corresponding dendrograms (b) in prion-diseased mice and age-matched controls. Note that aging seemed to shrink astrocyte arbors and that prion disease seemed to be associated with thicker astrocyte processes and larger cell bodies. EE was associated with an increase in the number of branches and a reduction in the prion disease-induced hypertrophy. Linear dendrogram of each astrocyte arbor with the length of each branch segment is displayed to scale as vertical lines and sister branches is horizontally displaced. Arrows indicate changes induced by each variable (environment, infection, and age). Dendrogram was plotted and analyzed with Neuroexplorer (MicroBrightField). Branches of the same parental (primary branch) trunk are shown in one color. Scale bar: 10 μm.

Figure 9

Graphic representations of convex-hull analysis showing that EE increased by as much as 91% the total tree field volume of the astrocytes from NBH-EE as compared with NBH-SE mice (^#p < 0.05; Bonferroni posttest).

Figure 10

Graphic representation of results of multivariate statistical analysis of morphometric features of all reconstructed astrocytes (n = 269). (a) Dendrogram illustrating astrocyte morphological phenotypes of the DG polymorphic layer from infected and control adults. Branch length, nodes, and soma area were the morphological features that most contributed to cluster formation in young adult groups. (b) Dendrogram illustrating astrocyte morphological phenotypes of the DG polymorphic layer from infected and control aged mice. Tree surface, soma area, branch length, branch nodes, and tree volumes were the morphological features that most contributed to the cluster formation of SE young adults and aged groups. (c) Canonical distribution of the discriminant analysis of morphological phenotypes of the polymorphic layer from both control and infected adult mice. Canonical analysis based on these morphological features revealed significant Mahalanobis distance between ME7 and NBH astrocytes and (e) between ME7-SE and all others. (d) Significant logarithmic correlation was detected between branch volumes and soma area, suggesting an interdependence between these morphological features.

Figure 11

Hierarchical cluster analysis of morphological features of astrocytes from the polymorphic layer limited to each experimental group. Astrocytes associated with SE exhibited only two morphological families, whereas those from EE animals showed four distinct morphological families. Astrocytes from ME7 groups were distributed into four different families with a larger surface area, and two of these astrocyte families exhibited significantly higher values for branch surface areas than all NBH families. Aged groups showed three astrocyte families. NBH-A formed a family with smaller surface area, suggesting that SE and aging, acting together, shrank astrocytes trees. In contrast, prion disease increased it. Branch surfaces (from all groups), soma area (from ME7 groups), and branch length (from EE and NBH-A groups) were the morphometric features that most contributed to cluster formation. (^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001, Bonferroni posttests).

Figure 12

Astrocyte morphological changes in prion-diseased adult and aged mice. (a) Cluster analysis and (b) canonical distribution of discriminant analysis. Note the clear distinction between NBH and ME7 young adults in both cluster and discriminant canonical analysis. Branch volume was the morphometric feature that most contributed to the cluster formation. However, ME7-A and NBH-EE control mice occupy the same cluster in the dendrogram, suggesting that prion disease in aged mice did not alter astrocyte morphology as it did in adult mice. (c) Soma volumes estimated by the nucleator method are linearly correlated with branch and volumes.