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

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

Effects of U0126 on enhanced vasoconstrictor receptor expression.

<p>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 (<b>A and D</b>), ischemia-induced rats treated with vehicle in (<b>B and E</b>) and for ischemia-induced rats treated with U0126 in (<b>C and F</b>). The upper row are showing red fluorescent staining with antibodies against ET_B receptor (<b>A-C</b>) and in the lower row photomicrographs showing green fluorescent staining with antibodies against 5-HT_1B receptor (<b>D-F</b>). 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.</p

Effects of U0126 on delayed neuronal cell death.

<p>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 (<b>A-C</b>). (<b>A</b>) TUNEL staining of coronal sections from sham-operated rats. (<b>B</b>) TUNEL positive cells in the vehicle treated group. (<b>C</b>) U0126 group. (<b>D</b>) 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. *<i>p</i><0.05.</p

Time-course of sensorimotor deficits.

<p>Rotating pole test (<b>A</b>) and grip strength test (<b>B</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. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001</p

Effects of U0126 on enhanced vasoconstriction.

<p>Graphs showing concentration-contraction curves for MCAs (<b>A and C</b>) and ACAs (<b>B and D</b>) stimulated with cumulative doses of ET-1 (<b>A and B</b>) or 5-CT (<b>C and D</b>) 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. ***<i>p</i><0.001</p

Physiological Parameters.

<p>The following physiological parameters were measured during surgery; mean arterial blood pressure (MABP), pH, CO₂ and O₂ pressure (p), body and cranial temperature (T) of control animals (sham) or animals subjected to global cerebral ischemia and reperfusion for 0, 1, 3, 5 or 7 days. Values are means ± SEM, n = 3–24 rats in each group.</p

Frequency and characteristics of immune-related thyroid adverse events in patients with resected stage III/IV melanoma treated with adjuvant PD-1 inhibitors: a national cohort study

PURPOSE: Immune-related thyroid adverse events (irTAEs) occur frequently following immune checkpoint inhibitor (ICI) therapy. The purpose of this study is to provide knowledge about the incidence, clinical timeline characteristics, associated factors of irTAEs, and potential impact on treatment efficacy in patients with melanoma receiving adjuvant ICI therapy.METHODS: A national multicenter retrospective cohort study of patients with resected stage III/IV melanoma treated with adjuvant PD-1 inhibitors between November 2018 and December 2020. Data were extracted from the Danish Metastatic Melanoma Database. The irTAEs were defined as two consecutive abnormal TSH values and subdivided into transient or persistent.RESULTS: Of 454 patients, 99 developed an irTAE (21.8%), of these were 46 transient (46.5%) and 53 persistent (53.5%). Median time to transient and persistent irTAE was 55 and 44 days, respectively (p = 0.57). A hyperthyroid phase followed by hypothyroidism was seen in 73.6% of persistent irTAEs, whereas 87% of transient irTAEs developed an isolated hypo- or hyperthyroid phase. Multiple variable analysis demonstrated an association between irTAE and female sex (HR 2.45; 95% CI 1.63-3.70; p &lt; 0.001), but no association with recurrence-free survival (HR 0.86; 95% CI 0.50-1.48; p = 0.587) or overall survival (HR 1.05; 95% CI 0.52-2.12, p = 0.891).CONCLUSIONS: IrTAE is a common side effect to PD-1 inhibitors primarily occurring within the first 3 months, with a high risk of persistency. Female sex is a strong predictive factor. IrTAE was not associated with improved clinical outcome.</p