Adrenomedullin prevents sex-dependent impairment of autoregulation during hypotension after piglet brain injury through inhibition of ERK MAPK upregulation

Abstract

Cerebrospinal fluid (CSF) adrenomedullin (ADM) levels are increased in female, but remain unchanged in male, piglets after fluid percussion injury (FPI) of the brain. Subthreshold vascular concentrations of ADM restore impaired hypotensive pial artery dilation after FPI more in males than females. Extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK) is upregulated and contributes to reductions in cerebral blood flow (CBF) after FPI. We hypothesized that ADM prevents sex-dependent impairment of autoregulation during hypotension after FPI through inhibition of ERK MAPK upregulation. FPI increased ERK MAPK more in males than in females. CBF was unchanged during hypotension in sham animals, was reduced more in males than in females after FPI during normotension, and was further reduced in males than in females during hypotension and after FPI. ADM and the ERK MAPK antagonist U 0126 prevented reductions in CBF during hypotension and FPI more in males than in females. Transcranial Doppler (TCD) blood flow velocity was unchanged during hypotension in sham animals, was decreased during hypotension and FPI in male but not in female pigs, and was ameliorated by ADM. Intracranial pressure (ICP) was increased after FPI more in male than in female animals. ADM blunted elevated ICP during FPI and hypotension in males, but not in females. ADM prevented reductions in cerebral perfusion pressure (CPP) during FPI and hypotension in males but not in females. The calculated autoregulatory index was unchanged during hypotension in sham animals, but was reduced more in males than females during hypotension and FPI. ADM prevented reductions in autoregulation during hypotension and FPI more in males than females. These data indicate that ADM prevented loss of cerebral autoregulation after FPI in a sex-dependent and ERK MAPK-dependent manner.

FIG. 1.

Typical sections of the parietal cortex from sham control, FPI, and FPI + ADM-treated newborn male and female piglets. (A) Uninjured sham control newborn male treated with vehicle and stained with rabbit anti-ADM human serum. (B) FPI newborn male treated with vehicle and stained with rabbit anti-ADM. (C) FPI newborn female treated with vehicle and stained with mouse anti-neuronal nuclei monoclonal antibody (5 μg/mL) as a neuronal marker along with rabbit anti-ADM. (D) Uninjured sham control newborn female treated with vehicle and stained with rabbit anti-ADM. (E) FPI newborn female treated with vehicle and stained with rabbit anti-ADM. (F) FPI newborn female vehicle stained with mouse IgG as a negative control. (G) Sham control newborn male treated with vehicle and stained with anti-phospho-ERK rabbit monoclonal antibody (2 μg/mL). (H) FPI newborn male vehicle-treated stained with anti-phospho-ERK rabbit monoclonal antibody. (I) FPI newborn male ADM (0.1 mg/kg IV) treated and stained with anti-phospho-ERK rabbit monoclonal antibody. (J) Sham control newborn female treated with vehicle and stained with anti-phospho-ERK rabbit monoclonal antibody. (K) FPI newborn female treated with vehicle and stained with anti-phospho-ERK rabbit monoclonal antibody. (L) FPI newborn female treated with ADM (0.1 mg/kg IV) and stained with anti-phospho-ERK rabbit monoclonal antibody. Original magnification is 100 × for all panels, except for inserts for which it is 400 × (n = 2 animals per group; FPI, fluid percussion injury; ADM, adrenomedullin; ERK, extracellular signal-related kinase).

FIG. 2.

Phosphorylation of ERK MAPK in CSF prior to FPI (0 time), and as a function of time after FPI, in pigs treated with vehicle (FPI), ADM (0.1 mg/kg IV) + FPI, ADM 22-52 (10⁻⁷ M) + FPI, or U 0126 (1 mg/kg IV) + FPI (n = 6). Time 0 is the time point immediately before injury. Data were analyzed by repeated measures ANOVA and expressed as percentage of control by ELISA determination of phospho ERK MAPK and total ERK MAPK isoforms, and subsequent normalization to the total form. (A) Male animals. (B) Female animals (*p < 0.05 compared with the corresponding value at time point 0; ⁺p < 0.05 compared with the corresponding vehicle-treated value; ^#p < 0.05 compared with the corresponding female value; FPI, fluid percussion injury; ADM, adrenomedullin; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay).

FIG. 3.

Influence of FPI on pial artery diameter in vehicle (FPI), ADM (10⁻¹⁰ M), ADM 22-52 (10⁻⁷ M), and U 0126 (10⁻⁶ M) pre-treated newborn male (A) and female (B) pigs (n = 6). Data were analyzed by repeated measures ANOVA (*p < 0.05 compared with the corresponding FPI vehicle value; ⁺p < 0.05 compared with the corresponding female value; FPI, fluid percussion injury; ADM, adrenomedullin; ANOVA, analysis of variance).

FIG. 4.

Influence of FPI on total CBF in vehicle (FPI), ADM (0.1 mg/kg IV), ADM 22-52 (0.1 mg/kg IV), or U 0126 (1 mg/kg IV) pre-treated newborn male (A) and female (B) pigs (n = 3). Time point 0 is the time immediately before injury. Data were analyzed by repeated measures ANOVA (*p < 0.05 compared with the corresponding time point 0 value; ⁺p < 0.05 compared with the corresponding vehicle pre-treated value; ^#p < 0.05 compared with the corresponding female value; CBF, cerebral blood flow; FPI, fluid percussion injury; ADM, adrenomedullin; ANOVA, analysis of variance).

FIG. 5.

Influence of moderate and severe hypotension on pial artery diameter before (time point 0) and after FPI in newborn male (A) and female (B) pigs pretreated with vehicle (FPI), ADM (0.1 mg/kg IV), ADM 22-52 (10⁻⁷ M), or U 0126 (1 mg/kg IV; n = 6). Data were analyzed by repeated measures ANOVA (*p < 0.05 compared with the corresponding time point 0 value; ⁺p < 0.05 compared with the corresponding vehicle-treated value; ^#p < 0.05 compared with the corresponding female value; FPI, fluid percussion injury; ADM, adrenomedullin; ANOVA, analysis of variance).

FIG. 6.

CBF in (A) parietal cortex and (B) hippocampus during control (C), moderate and severe hypotension (MH and SH) in sham, FPI, and FPI + ADM-treated (0.1 mg/kg IV) male and female pigs (n = 6). Data analyzed by repeated measures ANOVA (*p < 0.05 compared to the corresponding sham value; ⁺p < 0.05 compared to the corresponding control value; ^#p < 0.05 compared to the corresponding FPI value; ^&p < 0.05 compared to the corresponding male value; CBF, cerebral blood flow; FPI, fluid percussion injury; ADM, adrenomedullin; ANOVA, analysis of variance).

FIG. 7.

(A) Intracranial pressure (ICP) and (B) cerebral perfusion pressure (CPP) during control (C), and moderate and severe hypotension (MH and SH) in sham, FPI, and FPI + ADM-treated (0.1 mg/kg IV) male and female pigs (n = 6). Data were analyzed by repeated measures analysis of variance (ANOVA) (*p < 0.05 compared to the corresponding sham value in A, and compared to the corresponding C value in B; ⁺p < 0.05 compared to the corresponding FPI value in A, and compared to the corresponding sham value in B; ^#p < 0.05 compared to the corresponding male value in A, and compared to the corresponding FPI value in B; ^&p < 0.05 compared to the corresponding male value in B; FPI, fluid percussion injury; ADM, adrenomedullin).

FIG. 8.

Typical transcranial Doppler (TCD) tracings from paired groups of animals during normotension and severe hypotension (Sham-M, male sham controls; FPI-vehicle-M, FPI + vehicle males; FPI-ADM-M, FPI + ADM males; Sham-F, female sham controls; FPI-vehicle-F, FPI + vehicle females; FPI-ADM-F, FPI + ADM females; FPI, fluid percussion injury; ADM, adrenomedullin; P.I., peak interval, Dias., diastolic).

FIG. 9.

Autoregulatory index (ARI) values for moderate and severe hypotension (MH and SH) in sham male, FPI male, FPI + ADM male, sham female, FPI female, and FPI + ADM female animals (n = 4−5). Data were analyzed by repeated measures analysis of variance (ANOVA). (*p < 0.05 compared to the corresponding sham value; ⁺p < 0.05 compared to the corresponding FPI vehicle value; ^#p < 0.05 compared to the corresponding female value; FPI, fluid percussion injury; ADM, adrenomedullin).