Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients

Abstract

Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke.

Keywords: Gerotarget; aging; neurogenesis; post-stroke depression; serotonin receptor type B; stroke.

Figure 1. Experimental design and time course of spontaneous activity and hedonic behaviour after stroke

A. Schematic overview of the experimental design.B.: Before surgery, 79% of young animals (blue line) and 75% of old animals (red line) were hedonic. After stroke, the number of anhedonic animals increased to 42% by day 21 and to 57% by day 28. The fluoxetine treatment however did reduce anhedonic behaviour to 35% by day 28 (Figure 1B, red dashed line). ** p < .020.

Figure 2. Assessment of post-stroke depression in aged animals

A. After stroke, forced swimm cumulative activity decreased steadily form day 1 to day 28. Fluoxetine treatment, however, reversed partially the cumulative activity (dashed line). B. By behaviour activity type, swimming had a modest contribution to the total activity index. Stroke led to an impairment of climbing activity that reached the lowest value at day 21 post-stroke while immobility increased steadily from pre-surgery levels to day 21. The anti-depressant treatment led to an improvement in the swimming behaviour mostly (5-fold increase, p = 0.001) followed by an improvement in immobility behaviour (1.8-fold, p = 0.02).

Figure 3. Age-specific post-stroke up-regulation of Htr2B mRNA in the peri-infarct area of rats

A.-C. By day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. D. By RT-PCR at day 3 post-stroke there was a 15-fold up-regulation in the expression of Htr2b mRNA that was 7-fold higher in the lesioned area of aged animals than in young animals (N = 7/age group). Fluoxetine treatment, however, caused by day 28, a significant increase (1.9-fold) in the Htr2b mRNA levels in the perilesional area of aged rats E.. In the perinfarcted area of aged rats HTR2B-positive cells (green) were co-localized with NeuN-positive nuclei (red) at day3 after stroke in aged rat brains (arrows). F. Although the mRNA expression was largely down-regulated by day14 post-stroke, the HTR2B/NeuN co-localization persisted in the peri-infarcted area of aged rats (arrows).

Figure 4. Histochemical localization of HTRB2B in the peri-infarct area of human subjects

By chemical immunostaining, the HTR2B receptor in human subjects has a perinuclear localization (A., arrows; Vector blue-grey development) in the penumbral region of post-mortem stroke patients. By immunofluorescence, a similar co-localization was observed in the peri-infarcted area between HTR2B-positive cells (green) and cells labeled by beta III tubulin (red), a marker of mature neuronal cells (B., arrows). In addition to the neuronal co-localization, we also noted the co-localization with astrocytes labeled with FITC-GFAP (C., arrows) in the penumbral region of post-mortem stroke patients. However, there was no co-localization with astrocytes in the unlesioned, contralateral region of the same post-stroke patient (D., arrowheads). Bar, 100 μm. Abbreviations: PI, periinfarct.

Figure 5. HTR2B is co-localized with brain macrophages in the perinfarcted area of aged rats and human subjects

A. In the peri-infarcted area of young animals, activated microglia (arrowheads) was seen in close association with HTR2B⁺ neurons (arrows). The close proximity of HTR2B and brain microglia was less evident at day 14 post-stroke B.. C. In aged rats, numerous microglial cells immunopositive for IbaI were wrapped around neurons expressing HTR2B (arrowheads), suggesting a pre-phagocytic pathology. In addition, a robust co-localization of the microglia marker Iba1 with HTR2B-expressing blood vessels (inset; Bv). D. At day14, the co-localization between Iba1 and HtR2B was less evident in aged animals. E. In the peri-lesional area of human stroke victims we noted numerous phagocytic microglia expressing AIF1 (red), co-localized with HTR2B-expressing neurons (green). F. In remote areas with reference to the infarct core, numerous blood vessels were positive for HTR2B (green, arrows). Bar, 100 μm. Abbreviations: Bv, blood vessel; IC, infarct core; PI, periinfarct.

Figure 6. Fluoxetine stimulates post-stroke neurogenesis in aged animals

A.,D. Note the vigorous post-stroke neurogenesis as evidenced by an increased number of DCX⁺ cells in the aged brains of animals treated with fluoxetine (2.2-fold, p = 0.02) as compared to the modest expression in the brains of control animals B. so that the number of DCX⁺ in treated animals was almost similar to the number of DCX⁺ found in the subventricular zone (SVZ) of young animals ipsilateral to stroke C..