Exercise promotes axon regeneration of newborn striatonigral and corticonigral projection neurons in rats after ischemic stroke

Abstract

Newborn striatal neurons induced by middle cerebral artery occlusion (MCAO) can form functional projections targeting into the substantia nigra, which should be very important for the recovery of motor function. Exercise training post-stroke improves motor recovery in clinic patients and increases striatal neurogenesis in experimental animals. This study aimed to investigate the effects of exercise on axon regeneration of newborn projection neurons in adult rat brains following ischemic stroke. Rats were subjected to a transient MCAO to induce focal cerebral ischemic injury, followed by 30 minutes of exercise training daily from 5 to 28 days after MCAO. Motor function was tested using the rotarod test. We used fluorogold (FG) nigral injection to trace striatonigral and corticonigral projection neurons, and green fluorescent protein (GFP)-targeting retroviral vectors combined with FG double labeling (GFP(+) -FG(+)) to detect newborn projection neurons. The results showed that exercise improved the recovery of motor function of rats after MCAO. Meanwhile, exercise also increased the levels of BDNF and VEGF, and reduced Nogo-A in ischemic brain. On this condition, we further found that exercise significantly increased the number of GFP(+) -FG(+) neurons in the striatum and frontal and parietal cortex ipsilateral to MCAO, suggesting an increase of newborn striatonigral and corticonigral projection neurons by exercise post-stroke. In addition, we found that exercise also increased NeuN(+) and FG(+) cells in the striatum and frontal and parietal cortex, the ischemic territory, and tyrosine hydroxylase (TH) immunopositive staining cells in the substantia nigra, a region remote from the ischemic territory. Our results provide the first evidence that exercise can effectively enhance the capacity for regeneration of newborn projection neurons in ischemic injured mammalian brains while improving motor function. Our results provide a very important cellular mechanism to illustrate the effectiveness of rehabilitative treatment post-stroke in the clinic.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Design of animal experiments and neural function tests.

A: Animals were injected with GFP-labeled retrovirus 1 day before receiving MCAO, FG injection at 84 days and sacrificed at 91 days. All animals were given treadmill exercise before MCAO and the rats in MCAO+Ex group were given treadmill exercise after MCAO. B: Neuro-score was tested at 1, 3, 7 days after MCAO. C: Motor function was assessed using the rotarod test at different time points after MCAO as shown in C. The group which received exercise post-stroke expressed better behavioral performance in rotarod test. Data are shown as mean ± S.E.M. MCAO group (n = 8); MCAO+Ex group (n = 6). *P<0.05 vs MCAO group.

Figure 2. The fluorescent signal of FG and immunohistochemical signals of NeuN and FG in ischemic brain.

A: Photographs showed that FG was injected into the ipsilateral SN (a, b and c) and its signals were observed in the pyramidal shaped neurons in the ipsilateral frontal and parietal cortex (f) and striatum (g) at 7 days after injection. Photographs c, f and g were magnifications of the square in photos b and e. Arrowhead in photograph b indicated where the needle was inserted toward the SN. B: Histochemical photographs displayed NeuN+ and FG+ single-labeling cells in the striatum (left) and cortex (right) of ischemic rat brain at 13 weeks after MCAO. C: Quantification showed that exercise post-stroke increased the number of NeuN+ and FG+ cells in ischemic rat brain. Data are shown as mean ± S.E.M. MCAO group (n = 8); MCAO+Ex group (n = 6). *P<0.05 vs MCAO group. Scale bars are 1 mm in b and e; 100 µm in c, f and g; and 30 µm in B.

Figure 3. Newborn neurons formed projections to the substantia nigra in ischemic rat brain.

A–D: Confocal microphotographs showed GFP+-NeuN+-FG+ triple-labeling cells (triple arrowheads) in the ipsilateral striatum (A and B) and parietal cortex (C and D) at 13 weeks after MCAO. NeuN+ single-labeling cells, GFP+-NeuN+ and NeuN+-FG+ double-labeling cells were indicated by single arrowhead, double arrows and double arrowheads, respectively. Scale bars are 20 µm.

Figure 4. Increase of the formation of newborn projection neurons in adult ischemic brain by exercise post-stroke.

Double immunohistochemical staining of FG (blue) and GFP (brown) was used to detect newly generated projection neurons. A: Histochemical photographs showed GFP+-FG+ double staining cells as indicated by double red arrowheads. GFP+ and FG+ single-labeling cells were indicated by single red arrow and arrowhead, respectively. B: Quantitative analysis showed that exercise post-stroke significantly increased the number of newborn striato- and cortico-nigral projection neurons in ischemic rat brain. Data are shown as mean ± S.E.M. MCAO group (n = 8); MCAO+Ex group (n = 6). *P<0.05 vs MCAO group. Scale bar in A is 100 µm.

Figure 5. Effect of exercise on the expression of axonal growth associated protein in ischemic rat brain.

Brain protein samples from rats subjected to MCAO for 30: Western blot results and corresponding densitometric analysis showed that the levels of BDNF, SYN and VEGF were increased in MCAO+Ex group compared with MCAO group; and that Nogo-A levels were reduced in the MCAO+Ex group. C: Correlation results showed that SYN levels were increased after exercise and were negatively correlated with Nogo-A levels in ischemic rat brain. Data are shown as mean ± S.E.M. *P<0.05 vs MCAO group; MCAO group (n = 4); MCAO+Ex group (n = 5).

Figure 6. Effect of exercise on tyrosine hydroxylase expression in adult ischemic brain.

A: Histochemical photographs showed TH+ cells in ipsilateral SN at 13 weeks after MCAO. B: Quantity analyzing data demonstrated that exercise post-stroke increased the number of TH+ cells in the SN. C and D: Western blot and quantitative data showed that exercise post-stroke had no significant effect on TH protein expression in the ipsilateral striatum. Data are shown as mean ± S.E.M. MCAO group (n = 4); MCAO+Ex group (n = 5). *P<0.05 vs MCAO group; Scale bar in A is 100 µm.