Lesion size-dependent synaptic and astrocytic responses in cortex contralateral to infarcts in middle-aged rats

Abstract

In young adult rats, unilateral lesions of the sensorimotor cortex lead to neuronal structural plasticity and synaptogenesis in the contralateral motor cortex, which is connected to the lesion site by transcallosal fibers. The contralesional neural plasticity varies with lesion size and results from the convergence of denervation-induced reactive plasticity and behavioral asymmetries. It was unknown whether similar effects occur in older animals. Furthermore, the coordination of synaptic responses with that of perisynaptic astrocytes had not been investigated. In this study, middle-aged rats (14-16 months old) were given sham-operations or unilateral ischemic lesions of the sensorimotor cortex. Fifty days later, numerical densities of neurons and synapses and morphological characteristics of astrocytic processes in layer V of the contralesional motor cortex were measured using stereological light and electron microscopy methods. Lesions resulted in behavioral asymmetries, but no significant synapse addition in the contralesional motor cortex. Synapse number per neuron was negatively correlated with lesion size and reduced opposite larger lesions compared with smaller ones. Astrocytic changes were also lesion size-dependent. Astrocytic hypertrophy was observed only after smaller lesions and was associated with greater coverage and greater numbers of synapses. These findings are consistent with those in younger rats indicating an inverse relationship between lesion size and adaptive neuronal restructuring in denervated cortex. However, they indicate that the synaptogenic reaction to this lesion is relatively limited in older animals. Finally, the results indicate that structural plasticity of perisynaptic astrocytes parallels, and could play a role in shaping, synaptic responses to postischemic denervation.

Fig. 1

Axodendritic synapses (white arrows) were identified in transmission electron micrographs (A, scale bar=500nm). Synapse number per neuron was negatively correlated with lesion size (B). A median split divided the lesion group into two subgroups. Representative Nissl stained coronal sections and lesion reconstructions are shown for the two lesion sizes (C). The open rectangle in the contralesional cortex of the photomicrographs indicates the layer V sample region for the synaptic and neuronal density estimates. Numbers are coordinates in mm relative to bregma. Scale bar = 1 mm. In C, lesion reconstructions from different animals are overlaid so that the darker color indicates greater overlap of lesion territory between brains. In the Schallert cylinder test (D), the Large Lesion group had significantly reduced use of the impaired limb compared to the Shams (*p<0.05).

Fig. 2

Neuropil volume per neuron (A) and synapse number per neuron (B) in the contralesional motor cortex and in one hemisphere of sham operates. Both neuropil volume per neuron and synapse number were significantly decreased in the Large Lesion group compared to the Small Lesion group (*p<0.05). Neither lesion group was significantly different from Sham.

Fig. 3

Fluoro-Jade B (FJB) staining for degenerating neurons in the contralesional (Contra), ipsilesional (Ipsi) and sham-operated (Sham) cortices. The open rectangle in each lower magnification image (A–D, I) indicates the region shown in the accompanying higher magnification image (E–H, J). FJB (+) neurons were abundant in the peri-infarct area on post-lesion day 1 (B and F). They were less abundant by post-lesion day 3 (D and H). Clearly labeled neurons were rarely found in either the contralesional cortex (E and G) or in sham-operates (J). In quantitative analyses (K), there were significantly more FJB (+) neurons in the peri-infarct region at each time point compared with the contralateral side (*p<0.001 vs. Contra). There were significantly fewer FJB+ cells in peri-infarct cortex at Day 3 compared with Day 1 (†p <0.001 vs. Day 1 Ipsi). There was no significant difference in the contralesional cortex between two time points.

Fig. 4

Astrocytic hypertrophy in the contralesional homotopic cortex. Astrocytic processes (asterisks) were identified in electron micrographs (A, scale bar=500nm). Increased astrocytic process volume per neuron was observed only in the Small Lesion group compared with Sham (B, *p<0.05, †p<0.01).

Fig. 5

Correlations between astrocytic hypertrophy and synapse numbers in the contralesional homotopic cortex. Astrocytic process volume per neuron was significantly correlated with synapse number per neuron in the Lesion group (A, p < 0.01), but not in Sham (B).