Expressional changes in cerebrovascular receptors after experimental transient forebrain ischemia

Abstract

Background: Global ischemic stroke is one of the most prominent consequences of cardiac arrest, since the diminished blood flow to the brain results in cell damage and sometimes permanently impaired neurological function. The post-arrest period is often characterised by cerebral hypoperfusion due to subacute hemodynamic disturbances, the pathophysiology of which are poorly understood. In two other types of stroke, focal ischemic stroke and subarachnoid hemorrhage, it has earlier been demonstrated that the expression of certain vasoconstrictor receptors is increased in cerebral arteries several days after the insult, a phenomenon that leads to increased contraction of cerebral arteries, reduced perfusion of the affected area and worsened ischemic damage. Based on these findings, the aim of the present study was to investigate if transient global cerebral ischemia is associated with upregulation of vasoconstrictive endothelin and 5-hydroxytryptamine receptors in cerebral arteries. Experimental transient forebrain ischemia of varying durations was induced in male wistar rats, followed by reperfusion for 48 hours. Neurological function was assessed daily by three different tests and cerebrovascular expression and contractile function of endothelin and 5-hydroxytryptamine receptors were evaluated by wire myography, immunohistochemistry and western blotting.

Results: Transient forebrain ischemia induced neurological deficits as well as functional upregulation of vasoconstrictive ET(B) and 5-HT(1B) receptors in cerebral arteries supplying mid- and forebrain regions. No receptor upregulation was seen in arteries supplying the hindbrain. Immunohistochemical stainings and western blotting demonstrated expressional upregulation of these receptor subtypes in the mid- and forebrain arteries and confirmed that the receptors were located in the smooth muscle layer of the cerebral arteries.

Conclusions: This study reveals a new pathophysiological aspect of global ischemic stroke, namely expressional upregulation of vasoconstrictor receptors in cerebral arteries two days after the insult, which might contribute to cerebral hypoperfusion and delayed neuronal damage after cardiac arrest.

Figure 1. Cerebral blood flow during transient forebrain ischemia induction.

Cortical cerebral blood flow (CBF) in the anterior part of the brain was measured before, during and after the 15 minutes transient forebrain ischemia with a laser-Doppler probe. The level of CBF before the ischemia (CBF baseline) was set to 100%. The two arrows depict the start and the end of the ischemic insult. Data are presented as means ± SEM (n = 2) in percentage of baseline CBF values.

Figure 2. Neurological outcome.

Bar graphs showing mean (± SEM) rotating pole scores (A and B) and grip strengths (C and D) for control-operated rats (sham) and ischemia-induced rats subjected to 15, 10 and 2 minutes of transient forebrain ischemia (15 min, 10 min, and 2 min, respectively). Asterisks indicate significant difference between sham and ischemia groups. (A and B) Rotating pole scores were assessed on day 1 (A) and day 2 (B) after surgery. The horizontal pole was static (no rotation) or rotating at 3 rpm or 10 rpm. Statistically significant differences were assessed by 1-way ANOVA followed by Bonferroni’s multiple comparison tests. For each rotation speed n = 3–7 rats in each group. (C and D) Grip strength was measured in gram (g) using a grip strength meter on day 1 (C) and day 2 (D) after surgery. Statistically significant differences were determined using 1-way ANOVA, n = 7–8 rats in each group. (E) Muscle strength was measured in seconds (s) rats managed to hang in a grid where highest score was set to 30 seconds. Statistical differences between sham and ischemia groups were determined using student’s t-test, n = 2–6 rats in each group. *p<0.05, **p<0.01 and ***p<0.001.

Figure 3. Contractile function of endothelin receptors in cerebral arteries.

Graphs showing concentration-contraction curves elicited by the cumulative application of endothelin-1 (ET-1) to basilar artery (BA) (A), middle cerebral artery (MCA) (B) and anterior cerebral artery (ACA) (C) segments from control-operated rats (sham) and rats subjected to 15, 10 or 2 minutes of transient forebrain ischemia (15 min, 10 min, and 2 min, respectively). (D) Shows contractile responses to the ET_B-specific agonist sarafatoxin 6c (S6c) in ACA segments from sham-operated rats and rats subjected to 10 or 15 minutes of transient forebrain ischemia. Values are expressed as means ± SEM in percentage of contractions evoked by 63 mM of K+. Significant differences between sham and 15 minutes ischemia are determined by 2-way ANOVA followed by Bonferroni’s posttest indicated by the asterisks on the right side respective above the curves.

Figure 4. Contractile function of 5-HT_1B receptors in cerebral arteries.

Graphs showing concentration-contraction curves elicited by the cumulative application of 5-carboxamidotryptamine (5-CT) to basilar artery (BA) (A), middle cerebral artery (MCA) (B) and anterior cerebral artery (ACA) (C) segments from control-operated rats (sham) and rats subjected to 15, 10 or 2 minutes of transient forebrain ischemia (15 min, 10 min, and 2 min, respectively). (D) Shows contractile response to 5-CT in MCA segments from sham-operated rats and rats subjected to 15 minutes of ischemia, in the presence and absence of the selective 5-hydroxotryptamin type 1B receptor (5-HT_1B) antagonist GR55562. Values are expressed as means ± SEM in percentage of contractions evoked by 63 mM of K+. Significant differences between sham and 15 minutes ischemia are determined by 2-way ANOVA followed by Bonferroni’s posttest indicated by the asterisks on the right side respective above the curves.

Figure 5. Endothelin receptor immunohistochemistry.

Photomicrographs showing immunohistochemical stainings of anterior cerebral artery (ACA) sections with antibodies against endothelin type B (ET_B) (A and B) or endothelin typ A (ET_A) (D and E) receptors. Bar graphs show quantifications of staining intensities for ET_B (C) and ET_A (F) receptors. Two sections from each of 6 to 11 animals were analyzed. Data are presented as mean SEM in percentages of the mean staining intensity in control-operated (sham) animals. Significant differences between sham-operated and 15 minutes ischemia rats were determined using student´s t-test. *p< 0.05.

Figure 6. 5-HT receptor immunohistochemitry.

Photomicrographs showing immunohistochemical stainings of anterior cerebral artery (ACA) sections with antibodies against 5-hydroxotryptamin type 1B (5-HT_1B) (A and B) or 5-hydroxotryptamin type 2A (5-HT_2A) (D and E) receptors. Bar graphs show quantifications of staining intensities for 5-HT_1B (C) and 5-HT_2A (F) receptors. Two sections from each of 6 to 12 animals were analyzed. Data are presented as mean ± SEM in percentages of the mean staining intensity in control-operated (sham) animals. Significant differences between sham-operated and 15 minutes ischemia rats were determined using student’s t-test.

Figure 7. ET_B and 5-HT_1B receptor expression determined by western blotting.

Representative western blots and band intensity quantifications showing endothelin type B (ET_B) protein expression in basilar artery (BA) (A) and in pooled middle cerebral artery (MCA) and anterior cerebral artery (ACA) tissue (B), and 5-hydroxotryptamin type 1B (5-HT_1B) protein expression in BA (C) and in pooled MCA and ACA tissue (D) from control-operated (sham) rats and rats subjected to 15 minutes of transient forebrain ischemia. The band recognised by the ET_B receptor antibody was approximately 48 kDa and the band recognised by the 5-HT1B receptor antibody was approximately 41 kDa. Actin was used as a loading control. Data are expressed as mean ± SEM percentages of the mean band intensity in sham-operated animals and student’s t-test was used for statistical comparison. n = 5–13. *p<0.05.

Figure 8. Summary of data for neurological function, contractile function and protein expression levels of cerebrovascular ET_B and 5-HT_1B receptors as a function of the duration of the ischemic insult.

Data for endothelin type B (ET_B) and 5-hydroxotryptamin type 1B (5-HT_1B) receptor-mediated contractile function represent E_max values for the first phase (E_max-1) of the biphasic concentration-contraction curves for anterior cerebral arteries (ACA) stimulated with endothelin-1 (ET-1) and 5-carboxamidotryptamine (5-CT), respectively. Data for ET_B and 5-HT_1B protein expression levels represent receptor subtype-specific immunohistochemical staining intensities in ACAs. Neurology score data represents pooled data from grip strength and rotating pole. All data are values for ischemia-induced rats (2, 10 or 15 minutes) terminated 48 hours after ischemia in percentages of corresponding values for control-operated (sham) rats. All data are expressed as mean ± SEM.