Compromised blood-brain barrier competence in remote brain areas in ischemic stroke rats at the chronic stage

Abstract

Stroke is a life-threatening disease leading to long-term disability in stroke survivors. Cerebral functional insufficiency in chronic stroke might be due to pathological changes in brain areas remote from the initial ischemic lesion, i.e., diaschisis. Previously, we showed that the damaged blood-brain barrier (BBB) was involved in subacute diaschisis. The present study investigated BBB competence in chronic diaschisis by using a transient middle cerebral artery occlusion (tMCAO) rat model. Our results demonstrated significant BBB damage mostly in the ipsilateral striatum and motor cortex in rats at 30 days after tMCAO. The BBB alterations were also determined in the contralateral hemisphere via ultrastructural and immunohistochemical analyses. Major BBB pathological changes in contralateral remote striatum and motor cortex areas included 1) vacuolated endothelial cells containing large autophagosomes, 2) degenerated pericytes displaying mitochondria with cristae disruption, 3) degenerated astrocytes and perivascular edema, 4) Evans blue extravasation, and 5) appearance of parenchymal astrogliosis. Discrete analyses of striatal and motor cortex areas revealed significantly higher autophagosome accumulation in capillaries of ventral striatum and astrogliosis in dorsal striatum in both cerebral hemispheres. These widespread microvascular alterations in ipsilateral and contralateral brain hemispheres suggest persistent and/or continued BBB damage in chronic ischemia. The pathological changes in remote brain areas likely indicate chronic ischemic diaschisis, which should be considered in the development of treatment strategies for stroke.

Keywords: BBB; MCAO; astrocytes; autophagosomes; chronic diaschisis; rats.

Figure 1

Electron microscope examination of microvasculature in the rat striatum 30 days after tMCAO. Control (A–C): Representative areas of control rat striatum were characterized by the normal ultrastructural appearance of capillaries, motor neurons (N), neuropil, and myelinated axons (A). A single layer of endothelial cells (ECs) was surrounded by a single layer of basement membrane (BM) and enclosed by additional pericyte (P) cytoplasm, forming an intact BBB. Astrocyte (Ast) processes surrounding capillaries showed a normal morphology. tMCAO 30 days Ipsilateral hemisphere (D–F). D: In the hemisphere ipsilateral to tMCAO insult, ultrastructural abnormalities were observed in striatum capillary endothelia. ECs showed formation of autophagosomes (Aph) in their cytoplasm, with some autophagosomes extending from lumen to basal lamina in attenuated portions of the cells. Pericytes appeared swollen or completely degenerated. In close proximity to the capillary was a large protein-filled area created by degenerating astrocyte cell processes. Edematous astrocyte with lipofuscin inclusions was noted. E: Numerous large vacuoles were seen in EC cytoplasm (arrowheads) and perinuclear membrane separation was occasionally observed in EC. Pericyte was also swollen and contained enlarged mitochondria with disruption of cristae. Two large vacuoles (V) occupy cytoplasm of astrocyte. Collagen (Coll) formation surrounding capillary was detected. Myelin sheets in many axons were separated and disrupted (black A). Two degenerated axons (white A) showed evidence of disrupted axoplasmic transport. F: In ipsilateral striatum areas, edematous space between astrocyte, oligodendrocyte (Oligo), and neuron was noticed (*). Neuron also demonstrated swollen and edematous cytoplasm. tMCAO 30 days Contralateral hemisphere (G–I). G: In contralateral striatum, capillaries contained vacuolated EC with large autophagosomes. A degenerated pericyte occupied by mitochondria (m) with loss of cristae was also apparent. Surrounding the capillary were degenerated astrocytes (*) showing severe edema and free floating enlarged mitochondria. H: A capillary with vacuolization in EC cytoplasm in addition to the perinuclear membrane separation (arrowheads) proceeded to almost rupture the cell membrane, was determined. Adjacent to the capillary is an edematous perivascular space (*) containing enlarged mitochondria. Profile of dilated endoplasmic reticulum (ER) was observed in neuron cytoplasm near capillary. I: Another neuron with dilated nucleus appeared in contralateral striatum. Entirely degenerated astrocyte containing vacuoles and lipofuscin inclusions (Lf) was observed in close proximity to damaged neuron. En, endothelial cell; P, pericyte; BM, basement membrane; Ast, astrocyte; E, erythrocyte; Tj, tight junction; m, mitochondrion; A, axon; A (white), disrupted axoplasmic transport; V, vacuole; N, neuron; Nu, nucleus; Aph, autophagosome; ER, swollen endoplasmic reticulum; Lf, lipofuscin inclusion; Coll, collagen; Oligo, oligodendrocyte; arrowheads in E and H indicate separation of luminal EC membrane; arrowheads in I indicate dilated nucleus in neuron. Asterisks in D, E, F, G, and H indicate extracellular edema. Scale bar = 2 µm in A–E and F–I and = 500 nm in G.

Figure 2

Electron microscope examination of microvasculature in the rat motor cortex 30 days after tMCAO. Control (A–C). A,B: Similarly to striatum, capillaries in control motor cortex showed normal ultrastructure consisting of microvessels with a single endothelium layer (En), surrounded by BM, and partially bounded by pericyte (P) cytoplasm. Astrocyte (Ast) cell processes were adjacent to the outer capillary surface. Myelinated axons (A) were present and mitochondria showed a normal pattern. C: In control motor cortex areas, neurons (N), oligodendrocytes (Oligo), and astrocytes proximal to capillary demonstrated normal morphology. tMCAO 30 days Ipsilateral hemisphere (D–F). D: In the hemisphere ipsilateral to tMCAO damage, edematous EC with swollen mitochondria were adjacent to healthy ECs in the lumen. Astrocyte cell processes surrounding capillary demonstrated dilated endoplasmic reticulum (ER). E: Completely degenerated astrocyte with edematous cytoplasm was adjacent to capillary. Surrounding the capillary were cell processes of a degenerated pericyte with dilated endoplasmic reticulum and lipid drops (L). E,F: There was evidence of extracellular edema and myelin degeneration. F: Capillary EC displayed autophagosome (Aph) formation. Neuron appeared with swollen nucleus near capillary. tMCAO 30 days Contralateral hemisphere (G–I). G: In motor cortex of the hemisphere contralateral to tMCAO insult, a capillary was observed with swollen endothelium and complete pericyte degeneration with lipofuscin inclusions. H: In another capillary, a swollen pericyte contained enlarged mitochondria (m) with disruption of the cristae in addition to EC containing an autophagosome. Large areas of extracellular edema were observed surrounding the capillary (*). The thickness of endothelium was reduced in the area of perivascular edema. Microglia were detected adjacent to capillary and degenerated myelin. I: Edematous spaces (*) were revealed in neurons near nucleus or in their cytoplasm proximal to extracellular edema surrounding capillary. En, endothelial cell, P, pericyte; BM, basement membrane; Ast, astrocyte; E, erythrocyte; m, mitochondrion; A, axon; V, vacuole; N, neuron; Nu, nucleus; Aph, autophagosome; ER, swollen endoplasmic reticulum; L, lipid drop; Lf, lipofuscin inclusion; Oligo, oligodendrocyte; D, dendrite; right arrowhead in d indicates edematous EC with swollen mitochondria. Asterisks in E, F, G, H, and I indicate extracellular edema. Scale bar = 2 µm in B-G and I and = 500 nm in A, H.

Figure 3

Quantitative analysis of Evans Blue extravasation into the rat brain parenchyma and dye concentration in sera 30 days after tMCAO. A: Quantitative measurement of cerebral tissue EB content showed significantly (p<0.0001) higher extravasated EB levels in ipsilateral and contralateral hemispheres vs. control. Significantly (p=0.0047) elevated EB level was determined in ipsilateral hemisphere compared to contralateral. B: There was no significant difference in EB concentration in sera from tMCAO rats vs. controls.

Figure 4

Immunohistochemical analysis of Beclin-1 immunoexpression in capillary endothelium in the rat brain 30 days after tMCAO. Control Striatum Ipsi/Contralateral hemisphere (A–D): Immunofluorescent staining for Beclin-1 (green, arrowheads) showed typical expression in capillary endothelium of medial (A), lateral (B), dorsal (C), and ventral (D) striatum of control rats. tMCAO 30 days, Striatum Ipsilateral hemisphere (E–H): In the ipsilateral hemisphere, extensive Beclin-1 immunoexpression, indicating autophagosome accumulation within endothelium in numerous striatum capillaries in all analyzed areas, was observed. tMCAO 30 days, Striatum Contralateral hemisphere (I-L): In the contralateral striatum, higher Beclin-1 expression was determined in lateral (J) and ventral (L) areas compared to medial (I) or dorsal (K). In both ipsilateral (E–H) and contralateral (I-L) striatal capillaries, EB leakage (red, asterisks) was seen. Motor cortex (M–R): In motor cortex, increased Beclin-1 immunoexpression in capillary endothelium was determined in M2 (O) and M1 (P) areas in ipsilateral hemisphere vs. control (M,N). Q: Elevated Beclin-1 immunopositivity was noted in M2 contralateral motor cortex. R: Beclin-1 immunoexpression in M1 contralateral motor cortex was slightly elevated compared to M1 control (N). EB leakage (red, asterisks) was higher in M1 ipsilateral motor cortex area (P). M: Of note, EB dye in ipsilateral or contralateral hemispheres of controls was observed within capillary lumen (#). Striatum: medial (A,E,I), lateral (B,F,J), dorsal (C,G,K), and ventral (D,H,L) areas. Motor cortex: control ipsi/contralateral hemisphere (M – M2 area, N – M1 area), tMCAO 30 days ipsilateral hemisphere (O – M2 area, P – M1 area), and tMCAO 30 days contralateral hemisphere (Q – M2 area, R – M1 area). Scale bar = 25 µm in A–R.

Figure 5

Quantitative analysis of Beclin-1 immunoexpression in capillary endothelium in the rat brain 30 days after tMCAO. A: Schematic representation of analyzed areas in the striatum and motor cortex in the ipsilateral and contralateral hemispheres. B: Significant (p<0.0001) upregulation of Beclin-1 immunoexpression was detected in ipsilateral lateral (L), dorsal (D), and ventral (V) striatum areas of post-stroke rats compared to controls. In contralateral striatum, significant (p<0.0001) increase of Beclin-1 fluorescent expression was determined in area V, although elevation of protein expression was demonstrated in medial (M), L, and D areas vs. controls. C: In motor cortex, significant increase of Beclin-1 immunoexpression in capillary endothelium was determined in M2 (p=0.0005) and M1 (p<0.0001) areas in the ipsilateral hemisphere. Overexpression of Beclin-1 in contralateral motor cortex was noted in M2 area. More extensive Beclin-1 upregulation showed in capillary ECs of ipsilateral M1 (p=0.0012) than in M2 and in comparison to contralateral M2 (p=0.0004) and M1 (p<0.0001) motor cortex areas.

Figure 6

Immunohistochemical analysis of GFAP immunoexpression in the rat brain 30 days after tMCAO. Control Striatum Ipsi/Contralateral hemisphere (A–D): Normal appearance of parenchymal GFAP positive cells with well-defined astrocytes surrounding capillaries (green, arrowheads) was seen in all examined striatal areas of ipsilateral or contralateral hemisphere in control rats. EB dye was visible as small red dots (#) attached to the capillary lumen. tMCAO 30 days, Striatum Ipsilateral hemisphere (E–H): In the ipsilateral hemisphere of tMCAO rats, increased GFAP immunoreactivity, indicating astrogliosis, was determined in all analyzed striatal areas. tMCAO 30 days, Striatum Contralateral hemisphere (I–L): Similar, but less intensive, GFAP immunoreactivity was observed in contralateral striatum. EB capillary leakage was mostly detected in lateral and dorsal areas of ipsilateral (F,G, red, asterisks) and contralateral (J,K, red, asterisks) striatum. Motor cortex (M–R): In motor cortex, GFAP immunoexpression in M2 and M1 areas in ipsilateral (O,P) and contralateral (Q,R) hemispheres of tMCAO rats did not differ from controls (M,N). Importantly, dissociation of astrocytes from capillary lumen was observed in the striatum (F–H,J–L) and motor cortex (O–R) of both hemispheres in tMCAO rats. Striatum: medial (A,E,I), lateral (B,F,J), dorsal (C,G,K), and ventral (D,H,L) areas. Motor cortex: control ipsi/contralateral hemisphere (M – M2 area, N – M1 area), tMCAO 30 days ipsilateral hemisphere (O – M2 area, P – M1 area), and tMCAO 30 days contralateral hemisphere (Q – M2 area, R – M1 area). Scale bar = 50 µm in A–R.

Figure 7

Quantitative analysis of GFAP immunoexpression in the rat brain 30 days after tMCAO. A: Schematic representation of analyzed areas in the striatum and motor cortex in the ipsilateral and contralateral hemispheres. B: In ipsilateral striatum of tMCAO rats, significantly (p<0.0001) elevated GFAP immunoexpression was determined in all analyzed areas (M, L, D, and V) vs. controls. Only significant (p=0.0002) increase of GFAP immunoreactivity was determined in area D of contralateral striatum. C: There were no significant differences between tMCAO and control rats in GFAP immunoexpression in M2 and M1 motor cortices in ipsilateral or contralateral hemisphere, although elevation of GFAP immunoexpression was determined in ipsilateral M2 area of tMCAO rats. Also, ipsilateral M2 area of motor cortex was significantly (p=0.0009) higher than in contralateral side in tMCAO rats.