Early MEK1/2 inhibition after global cerebral ischemia in rats reduces brain damage and improves outcome by preventing delayed vasoconstrictor receptor upregulation

Abstract

Background: Global cerebral ischemia following cardiac arrest is associated with increased cerebral vasoconstriction and decreased cerebral blood flow, contributing to delayed neuronal cell death and neurological detriments in affected patients. We hypothesize that upregulation of contractile ETB and 5-HT1B receptors, previously demonstrated in cerebral arteries after experimental global ischemia, are a key mechanism behind insufficient perfusion of the post-ischemic brain, proposing blockade of this receptor upregulation as a novel target for prevention of cerebral hypoperfusion and delayed neuronal cell death after global cerebral ischemia. The aim was to characterize the time-course of receptor upregulation and associated neuronal damage after global ischemia and investigate whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and thereby improve functional outcome after global cerebral ischemia. Incomplete global cerebral ischemia was induced in Wistar rats and the time-course of enhanced contractile responses and the effect of U0126 in cerebral arteries were studied by wire myography and the neuronal cell death by TUNEL. The expression of ETB and 5-HT1B receptors was determined by immunofluorescence.

Results: Enhanced vasoconstriction peaked in fore- and midbrain arteries 3 days after ischemia. Neuronal cell death appeared initially in the hippocampus 3 days after ischemia and gradually increased until 7 days post-ischemia. Treatment with U0126 normalised cerebrovascular ETB and 5-HT1B receptor expression and contractile function, reduced hippocampal cell death and improved survival rate compared to vehicle treated animals.

Conclusions: Excessive cerebrovascular expression of contractile ETB and 5-HT1B receptors is a delayed response to global cerebral ischemia peaking 3 days after the insult, which likely contributes to the development of delayed neuronal damage. The enhanced cerebrovascular contractility can be prevented by treatment with the MEK1/2 inhibitor U0126, diminishes neuronal damage and improves survival rate, suggesting MEK1/2 inhibition as a novel strategy for early treatment of neurological consequences following global cerebral ischemia.

Figure 1. Time-course of enhanced cerebrovascular contractile responses after global cerebral ischemia.

Graphs summarizing concentration-contractions curves elicited by cumulative application of ET-1 (A) and 5-CT (B) to MCAs and ACAs from control rats (sham) or rats subjected to ischemia and reperfusion for 0, 1, 3, 5 or 7 days. The maximum contraction for E_max(1) and E_max(2) were calculated for each agonist at each post-ischemic day and normalized to the corresponding sham levels and the E_max value on the Y axis represents a mean value received from a composition of both E_max(1) and E_max(2). n = 3–15 rats in each group. Asterisks (* for MCA and # for ACA) indicate significant differences between sham and ischemia as determined by Student's t-test.

Figure 2. Time-course of sensorimotor deficits.

Rotating pole test (A) and grip strength test (B) was performed each day up to 7 days after sham-operation or global cerebral ischemia. All data are means ± SEM, n = 7–16 number of rats. Stars indicate significant differences between sham-operated rats compared to corresponding (same day after surgery) ischemia-operated rats as determined by Student's t-test. *p<0.05, **p<0.01, ***p<0.001

Figure 3. Time-course of neuronal cell death after global cerebral ischemia.

Representative microphotographs of HE staining (3A) and apoptotic DNA fragmentation detected by TUNEL staining (3B) in hippocampus CA1 area from sham operated rats or ischemia induced rats at 1, 3, 5 or 7 days of reperfusion. (3C) shows microphotographs of apoptotic DNA fragmentation detected by TUNEL staining in cortex from sham-operated and ischemia-induced rats 7 days after surgery. (3A and B) shows hippocampal CA1 neurons from sham-operated rat, ischemia-reperfusion after 1 day, ischemic hippocampal CA1 neurons from day 3 after surgery, ischemia rat at day 5 and hippocampal pyramidal cells from ischemia-induced rat a week after surgery. Scale bar (100 μm) in 3A (sham) applies to all figures (3A and B), magnification of 20x. (3C) shows frontal cortex area 3 (Fr3) from sham-operated rats, cingulate cortex area 2 (Cg2) from ischemia- induced rat, Fr3 from ischemia-induced rat, secondary motor cortex (M2) from ischemic rats and parietal cortex, posterior area, rostral part (PtPR) from ischemia-induced rats. Scale bar (100 μm) in 3C (sham) applies to all microphotographs from cortex, magnification of 20x. White arrows are pointing at TUNEL positive cells. n = 3 rats in each group.

Figure 4. Effects of U0126 on enhanced vasoconstriction.

Graphs showing concentration-contraction curves for MCAs (A and C) and ACAs (B and D) stimulated with cumulative doses of ET-1 (A and B) or 5-CT (C and D) obtained 3 days post-surgery. Data show sham-operated rats (sham), ischemia-induced rats treated with vehicle (ischemia + vehicle) and ischemia-induced rats treated with U0126 (ischemia + U0126) (30 mg/kg). Values are expressed as means ± SEM in percentage of contractions evoked by 63.5 mM of K⁺. Crosses indicate significant differences between sham and vehicle-treated ischemia-induced animals as determined by 2-way ANOVA. Stars indicate significant differences between U0126- and vehicle-treated ischemia induced rats as determined by 2-way ANOVA. n = 5–6 rats in each group. ***p<0.001

Figure 5. Effects of U0126 on enhanced vasoconstrictor receptor expression.

ET_B and 5-HT_1B receptor expression were determined by immunofluorescence staining in the smooth muscle cells of MCAs 3 days after surgery. Representative photomicrographs presented for sham-operated rats were shown in (A and D), ischemia-induced rats treated with vehicle in (B and E) and for ischemia-induced rats treated with U0126 in (C and F). The upper row are showing red fluorescent staining with antibodies against ET_B receptor (A-C) and in the lower row photomicrographs showing green fluorescent staining with antibodies against 5-HT_1B receptor (D-F). Three MCA sections from each group were analyzed and one representative image for each group is shown. n = 6-7 rats in each group. Scale bar in A applies to all figures, magnification of 40x.

Figure 6. Effects of U0126 on delayed neuronal cell death.

Representative microphotographs of TUNEL staining (apoptotic DNA fragmentation detection) show the effect of U0126 on the neuronal damage of hippocampal CA1 neurons, in rats 7 days after surgery (A-C). (A) TUNEL staining of coronal sections from sham-operated rats. (B) TUNEL positive cells in the vehicle treated group. (C) U0126 group. (D) Bars are illustrating quantitative measurements of U0126 effects on CA1 hippocampus neuronal apoptosis 7 days after ischemia, showing significant decreased number of TUNEL positive cells in U0126 compared to vehicle treated animals. Values are means ± SEM, n = 5–6 numbers of rats in each group. Significant differences were determined using Student's t-test. *p<0.05.

Figure 7. Effect of U0126 on outcome.

Graphs demonstrating neuronal observation of rat behavioral pattern (A) and survival rate (B) 1–7 days after rats were subjected to sham-operation or ischemia with either treatment of vehicle or U0126. (A) Ten different parameters of neurological observations were translated into scores, where 0 points indicated a normal behavioral pattern. *** on the right side indicate significant differences between vehicle and U0126 as determined by 1-way ANOVA. * above the error bar indicate significant differences between vehicle and U0126 determined by Student's t-test comparing rats from the same day. Values are means ± SEM, n = 5–11 numbers of rats in each group. *p<0.05, ***p<0.001. (B) Graph showing rat survival rate for ischemia-induced rats treated with either vehicle or U0126 up to a weak after induced ischemia. Rats in the U0126 group have a 100% survival rate whereas vehicle treated rats died in the subacute phase of ischemia, showing only 55% survival rate. n = 6–11 numbers of rats in each group.