uPA modulates the age-dependent effect of brain injury on cerebral hemodynamics through LRP and ERK MAPK

Abstract

We hypothesized that urokinase plasminogen activator (uPA) contributes to age-dependent early hyperemia after fluid percussion brain injury (FPI) by activating extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK), leading to histopathologic changes in the underlying cortex. Both cerebrospinal fluid (CSF) uPA and phosphorylation of CSF ERK MAPK was increased at 1 min after FPI in newborn pigs, but was unchanged in juvenile pigs. uPA and phosphorylated ERK MAPK, detectable in sham piglet brain by immunohistochemistry, was markedly elevated and associated with histopathology 4 h after FPI in the newborn but there was minimal staining and histopathology in the juvenile. EEIIMD, a peptide derived from PA inhibitor-1 that does not affect proteolysis, blunted FPI-induced phosphorylation of ERK MAPK. FPI produced pial artery dilation and increased cerebral blood flow at 1 min after insult in the newborn, but not in the juvenile. Antilipoprotein-related protein (LRP) antibody, EEIIMD, a soluble uPA antagonist, and the ERK MAPK antagonist U 0126 inhibited FPI-associated hyperemia. These data indicate that uPA is upregulated after FPI and produces an age-dependent early hyperemia followed by histopathology through an LRP- and ERK MAPK-dependent pathway.

Figure 1

Immunohistochemistry and histopathology 4 h after FPI. Sections of the parietal cortex from piglet brains after FPI (A–C, F, L, newborn vehicle; D, E, newborn-EEIIMD 1 mg/kg i.v.; G–I, juvenile-vehicle; J, K, juvenile-EEIIMD) and from uninjured control animal (M–O, newborn vehicle), were subjected to antigen retrieval in citrate buffer and stained with anti-phospho-ERK rabbit monoclonal antibody (2 μg/mL) (no. 4376; Cell Signaling) (A, D, G, J, M), or with anti-uPA monoclonal antibody (5 μg/mL) (no. 3689; American Diagnostica) (B, E, H, K, N), with mouse anti-neuronal nuclei monoclonal antibody (5 μg/mL) as a neuronal marker (no. MAB377; Chemicon International, Billerica, MA, USA) (C), or with nonimmune mouse IgG₁ as a negative control (F), secondary biotinylated anti-mouse IgG (1:200), followed by incubation with HRP-conjugated streptavidin. Magnification shown is ×400 for all panels except (I, L) which was ×100 and ×1,000 for inserts in (A, C). Adjacent sections from the same brains exposed to brain injury (I, L) and from uninjured control (O), were stained by H&E for histologic inspection. These data reflect an n of 2 per experimental group.

Figure 2

Influence of FPI on CSF uPA concentration (ng/mL) as a function of time after insult (min) in newborn and juvenile pigs, n=6. *P<0.05 versus corresponding pre-FPI (0 time) value ⁺ P<0.05 versus corresponding newborn value.

Figure 3

Phosphorylation of ERK MAPK in cortical periarachnoid CSF before FPI (0 mins), as a function of time (min) after FPI, and 4 h after FPI and additional exogenous administration of uPA (10⁻⁷ mol/L) in vehicle (FPI), EEIIMD (1 mg/kg i.v.)+ FPI, and U 0126 (1 mg/kg i.v.)+FPI animals, n=6. Data expressed as percentage of control by ELISA determination of phospho-MAPK and total MAPK isoforms and subsequent normalization to total form. (A) Newborn, (B) juvenile. *P<0.05 compared with corresponding 0 time value. ⁺P<0.05 compared with corresponding FPI non-pretreated value. ^#P<0.05 compared with corresponding 240 mins non-uPA-treated value. ^&P<0.05 compared with corresponding newborn value.

Figure 4

Influence of FPI on pial artery diameter in the absence and presence of EEIIMD (10⁻⁷ mol/L), suPAR (10⁻⁷ mol/L), anti-LRP antibody (Mab ag LRP, 10 μg/mL), and U 0126 (10⁻⁶ mol/L) pretreatment 30 mins before injury, n=6. (A) Newborn, (B) juvenile. *P<0.05 compared with corresponding FPI-alone value. ⁺P<0.05 compared with corresponding value in the newborn.

Figure 5

Blood flow in the cerebral cortex (CBF) in sham control, FPI, FPI+EEIIMD (1 mg/kg i.v.), FPI+suPAR (1 mg/kg i.v.), and FPI+anti-LRP antibody (0.1 mg/kg i.v.), 30 mins pretreatment animals, n=3 to 4. (A) newborn, (B) juvenile. *P<0.05 compared with corresponding sham control value. ⁺P<0.05 compared with corresponding FPI non-pretreated value. ^#P<0.05 compared with corresponding newborn value.