The MEK Inhibitor Trametinib Improves Outcomes following Subarachnoid Haemorrhage in Female Rats

Abstract

Aneurysmal subarachnoid haemorrhage (SAH) is a haemorrhagic stroke that causes approximately 5% of all stroke incidents. We have been working on a treatment strategy that targets changes in cerebrovascular contractile receptors, by blocking the MEK/ERK1/2 signalling pathway. Recently, a positive effect of trametinib was found in male rats, but investigations of both sexes in pre-clinical studies are an important necessity. In the current study, a SAH was induced in female rats, by autologous blood-injection into the pre-chiasmatic cistern. This produces a dramatic, transient increase in intracranial pressure (ICP) and an acute and prolonged decrease in cerebral blood flow. Rats were then treated with either vehicle or three doses of 0.5 mg/kg trametinib (specific MEK/ERK1/2 inhibitor) intraperitoneally at 3, 9, and 24 h after the SAH. The outcome was assessed by a panel of tests, including intracranial pressure (ICP), sensorimotor tests, a neurological outcome score, and myography. We observed a significant difference in arterial contractility and a reduction in subacute increases in ICP when the rats were treated with trametinib. The sensory motor and neurological outcomes in trametinib-treated rats were significantly improved, suggesting that the improved outcome in females is similar to that of males treated with trametinib.

Keywords: ET-1; SAH; cerebral artery; rat; subarachnoid haemorrhage; trametinib.

Figure 1

Inhibitory capacity of trametinib after 48 h of organ culture. (A) Maximal contraction to 60 mM K⁺ following incubation with 0.1, 1, and 10 µM of trametinib compared to vehicle. There is no significant difference between the vehicle and trametinib. (B) Concentration–response curves to the ET_B specific agonist S6c, following incubation with 0.1, 1, and 10 µM trametinib. Data are shown as mean ± SEM (n = 5).

Figure 2

Study outline. Overview of the procedures and analysis performed for the current study. All timepoints relate to time 0, which is the induction of the SAH.

Figure 3

Comparison of trametinib and vehicle treatment on contractility in the BA. (A) Maximal contraction to 60 mM K⁺ for the BAs. (B) Cumulative concentration–response curves to ET-1 (10⁻¹⁴–10⁻⁷ M) on the BA. Data are shown as mean ± SEM, with statistics calculated using two-way ANOVA followed by Bonferroni post-test, * p < 0.05.

Figure 4

Comparison of trametinib and vehicle treatment on contractility in the MCA (A) Cumulative concentration–response curves to ET-1 (10⁻¹⁴–10⁻⁷ M). (B) Cumulative concentration–response curves to 5-CT (10⁻¹²–3 × 10⁻⁵ M). Data are shown as mean ± SEM, with statistics calculated using two-way ANOVA followed by Bonferroni post-test, * p < 0.05.

Figure 5

Intracranial pressure post-SAH. The average ICP is significantly higher after 24 h in both groups. The increase in ICP is less in the rats treated with trametinib and, at 48 h, it is only significantly increased in the SAH + vehicle group. A multiple t-test with correction (* p < 0.05) was used for statistical analysis.

Figure 6

Weight loss and general well-being. (A) Animals were weighed at the starting point and after 24/48 h. There was a significant weight loss in both groups, with no significant effect of the trametinib treatment. (B) The general well-being ratings used a 22-point scoring system. Multiple t-tests with multiple testing correction (* p < 0.05) were used for statistical analysis.

Figure 7

Evaluation of neurologic function post-SAH. Rotating pole test at 10 rpm for: (A) 24 h post-SAH and (B) 48 h post-SAH. Scored with 4 counts per animal, i.e., 2 scores for left and right rotation, respectively; Low = Unable to traverse in one try; High = Able to traverse in one try. Fisher’s exact test (* p < 0.05) was used for statistics.