Chronic cerebral hypoperfusion induces vascular plasticity and hemodynamics but also neuronal degeneration and cognitive impairment

Abstract

Chronic cerebral hypoperfusion (CCH) induces cognitive impairment, but the compensative mechanism of cerebral blood flow (CBF) is not fully understood. The present study mainly investigated dynamic changes in CBF, angiogenesis, and cellular pathology in the cortex, the striatum, and the cerebellum, and also studied cognitive impairment of rats induced by bilateral common carotid artery occlusion (BCCAO). Magnetic resonance imaging (MRI) techniques, immunochemistry, and Morris water maze were employed to the study. The CBF of the cortex, striatum, and cerebellum dramatically decreased after right common carotid artery occlusion (RCCAO), and remained lower level at 2 weeks after BCCAO. It returned to the sham level from 3 to 6 weeks companied by the dilation of vertebral arteries after BCCAO. The number of microvessels declined at 2, 3, and 4 weeks but increased at 6 weeks after BCCAO. Neuronal degeneration occurred in the cortex and striatum from 2 to 6 weeks, but the number of glial cells dramatically increased at 4 weeks after BCCAO. Cognitive impairment of ischemic rats was directly related to ischemic duration. Our results suggest that CCH induces a compensative mechanism attempting to maintain optimal CBF to the brain. However, this limited compensation cannot prevent neuronal loss and cognitive impairment after permanent ischemia.

Figure 1

Magnetic resonance imaging (MRI) images showing changes in cerebral blood flow (CBF) of the parietal cortex (PC), striatum (ST), and cerebellum (CB). Color images are from 3D arterial spin labelling (ASL) and gray images from T2 MRI (A). Areas in red in ASL images represent the strongest signal of CBF; and areas in blue or green reflect the weakest signal of CBF; and the yellow color is intermediate signal between them. Pink or red circles indicate detected areas of CBF in the parietal cortex and striatum. Histogram showing quantitative results of CBF at different time points after bilateral common carotid artery occlusion (BCCAO) (B). CBF of bilateral parietal cortex, striatum, and cerebellum decreased at right common carotid artery occlusion (RCCAO), BCCAO, and 2 weeks after BCCAO, compared with pre-occlusion (P<0.01). But it increased in the right parietal cortex (P<0.05), right striatum (P<0.01) and both cerebellar hemispheres (P<0.01) compared with RCCAO group when BCCAO was induced. CBF of these three areas returned to the pre-occlusion level from 3 to 6 weeks after BCCAO. **P<0.01; ^#P<0.05; ^##P<0.01.

Figure 2

Changes in bilateral common carotid arteries (CCAs) and vertebral arteries (VAs) before and after bilateral common carotid artery occlusion (BCCAO). Three-dimensional time-of-flight MR angiography (3D TOF-MRA) images showing morphologic changes of CCAs and VAs (A). In pre-occlusion rats, bilateral CCAs were clearly seen (A-, arrows). After RCCA was occluded, its signal disappeared from the image (B-, arrow head), but LCCA was still visible (B-, arrow). After BCCAO, both CCA signals were absent from the image (C-, arrow head). At the same time, bead-like VAs were seen (C-, arrows). Gradual enlargement of VAs was observed from 2 to 6 weeks after BCCAO (D-, E-, and F-, arrows). Histogram showing changes in diameter and area of bilateral VAs (B, C). There was no difference in diameter and area of VAs between pre-occlusion group and right common carotid artery occlusion (RCCAO) group. The diameter of both VAs increased at 1 week after RCCAO when BCCAO was induced (P<0.01 versus RCCAO group in the left VA, P<0.01 versus the pre-occlusion and RCCAO groups in the right VA). VAs gradually became larger from 2, 3, and 4 weeks, and peaked at 6 weeks after BCCAO. Changes in VA area also underwent a similar pattern as that for VA diameter after BCCAO. **P<0.01; *P<0.05 compared with sham.

Figure 3

Morphologic changes in microvessels. Micrograph showing CD34 immunofluorescence staining of microvessels in the parietal cortex, striatum, and cerebellum at different points after bilateral common carotid artery occlusion (BCCAO) (A). In the sham group, stronger staining of CD34-positive microvessels was seen in the parietal cortex (A- and F, arrows), striatum (A1 and F1, arrows) and cerebellum (A2 and F2, arrows). However, the staining signal reduced at 2 weeks (B-, G, B1, G1, B2, G2) and 3 weeks (C-, H, C1, H1, C2, H2) in these three areas, and 4 weeks (D-, I, D1, I1) after BCCAO in the parietal cortex and the striatum. Stronger labelled CD34-positive microvessels reappeared earlier in the cerebellum at 4 weeks (D2, I2) and later at 6 weeks (E-, J, E1, J1, E2, J2) in the parietal cortex and the striatum after BCCAO. LPC, left parietal cortex; RPC, right parietal cortex; LST, left striatum; RST, right striatum; LCB, left cerebellum; RCB, right cerebellum. Histogram showing quantitative data (B–D): In the left parietal cortex, the percentage of the number and areas of microvessels decreased at 2, 3, and 4 weeks (P<0.01) and returned to the sham level at 6 wk (P>0.05). (B) In the right parietal cortex, the percentage of the number and areas of microvessels also decreased at 2, 3, and 4 weeks (P<0.01 and P<0.05) but increased at 6 weeks after BCCAO (RMVN, P<0.01 and RMVD, P<0.05) compared with the sham group. The percentage of the number and areas of microvessels in the striatum underwent a similar changing pattern (C). In the cerebellum, however, the percentage of the number and areas of microvessels decreased at 2 and 3 weeks but returned to the sham level from 4 weeks after BCCAO (D). LMVN, the number of microvessels in the left side; LMVD, the area of microvessels in the left side. RMVN, the number of microvessels in the right side; RMVD, the area of microvessels in the right side. **P<0.01; *P<0.05 compared with sham. Scale bar, 20 μm.

Figure 4

Changes in different types of cells in the parietal cortex. Micrograph showing changes of different types of cells in the parietal cortex at different points after bilateral common carotid artery occlusion (BCCAO) by immunofluorescent staining (A). NeuN-positive cells (neurons) were found in A1 to A5 (arrow), Iba1-positive cells (microglial cells) in B1 to B5 (arrowhead), and GFAP-positive cells (astrocytes) in C1 to C5 (arrow). The later two types cells were also seen in merge pictures of Iba1 and GFAP double immunostaining (D1 to D5). Histogram showing the results of quantitative analysis (B–D). The number of NeuN+ cells dramatically decreased to a half of sham level in bilateral parietal cortex (P<0.01) from 2 to 6 weeks after BCCAO (B). However, the numbers of Iba1+ cells did not significantly change at 2 and 3 weeks but significantly increased at 4 weeks, P<0.05 (L), P<0.01 (R), then returned to the normal level at 6 weeks after BCCAO (C). The number of GFAP+ cells decreased first at 2 weeks, then gradually increased from 3 weeks and reached peak values at 4 weeks (P<0.01) and 6 weeks (P<0.01) after BCCAO compared with sham (D). **P<0.01; *P<0.05; scale bar, 20 μm.

Figure 5

Changes in different types of cells in the striatum. Micrograph showing changes of different types of cells in the striatum at different points after bilateral common carotid artery occlusion (BCCAO) by immunofluorescent staining (A). NeuN-positive cells were found in A1 to A5 (arrow), Iba1-positive cells in B1 to B5 (arrow head), and GFAP-positive cells (astrocytes) in C1 to C5 (arrow). The later two types cells were also seen in merge pictures of Iba1 and GFAP double immunostaining (D1 to D5). Histogram showing the results of quantitative analysis (B–D). The number of NeuN-postive cells dramatically decreased to approximate 50% in bilateral striatum (P<0.01) from 2 to 6 weeks after BCCAO (B). The numbers of Iba1-positive cells in both striatum also dramatically declined at 2 weeks (P<0.01 at left and P<0.05 at right), then gradually increased and reached peak values at 4 and 6 weeks after BCCAO compared with sham (C). The number of GFAP-positive cells underwent a similar changing pattern. It first decreased at 2 weeks (P<0.01, D), then gradually increased and reached a peak value at 4 weeks (P<0.05) and then returned to the sham level at 6 weeks after BCCAO (P>0.05). **P<0.01; *P<0.05; scale bar, 20 μm.

Figure 6

Changes in thickness of granular layer of the cerebellum. (A) Micrograph showed the morphologic changes of granular layer of the cerebellum by HE staining (A1 to A5). Quantitative analysis showed that there was no difference in the thickness of granular layer in the cerebellum at different points after bilateral common carotid artery occlusion (BCCAO) (P>0.05, B), Scale bar, 20 μm.

Figure 7

Changes in memory behavior detection. Plot graph showing changes in escape latencies in all experimental groups (A). In day 1, the escape latency was significantly longer in all ischemic groups (P<0.01) compared with sham group. In day 2, longer escape latency was found at ischemic 6-week group (P<0.01). In day 3 and day 4, the escape latency was significantly prolonged at 2- and 4-week groups (P<0.05), and 6-week group (P<0.01) after bilateral common carotid artery occlusion (BCCAO). The travel path of rats crossing the platform (circle) or platform quadrant was shown in (B). Quantitative data showed that the frequency crossing the original platform was decreased in all ischemic groups (C). And the duration travelling in the original platform quadrant was also declined in ischemic groups (P<0.05 at 2 weeks, P<0.01 at 4 and 6 weeks after BCCAO) (D). **P<0.01; *P<0.05, compared with sham.