Inhibition of Myosin Light-Chain Kinase Attenuates Cerebral Edema after Traumatic Brain Injury in Postnatal Mice

Abstract

Traumatic brain injury (TBI) in children less than 8 years of age leads to decline in intelligence and executive functioning. Neurological outcomes after TBI correlate to development of cerebral edema, which affect survival rates after TBI. It has been shown that myosin light-chain kinase (MLCK) increases cerebral edema and that pretreatment with an MLCK inhibitor (ML-7) reduces cerebral edema. The aim of this study was to determine whether inhibition of MLCK after TBI in postnatal day 24 (PND-24) mice would prevent breakdown of the blood-brain barrier (BBB) and development of cerebral edema and improve neurological outcome. We used a closed head injury model of TBI. ML-7 or saline treatment was administered at 4 h and every 24 h until sacrifice or 5 days after TBI. Mice were sacrificed at 24 h, 48 h, and 72 h and 7 days after impact. Mice treated with ML-7 after TBI had decreased levels of MLCK-expressing cells (20.7 -4.8 vs. 149.3 -40.6), less albumin extravasation (28.3 -11.2 vs. 116.2 -60.7 mm 2 ) into surrounding parenchymal tissue, less Evans Blue extravasation (339 -314 vs. 4017 -560 ng/g), and showed a significant difference in wet/dry weight ratio (1.9 -0.07 vs. 2.2 -0.05 g), compared to saline-treated groups. Treatment with ML-7 also resulted in preserved neurological function measured by the wire hang test (57 vs. 21 sec) and two-object novel recognition test (old vs. new, 10.5 touches). We concluded that inhibition of MLCK reduces cerebral edema and preserves neurological function in PND-24 mice.

Role of myosin light chain and myosin light chain kinase in advanced glycation end product–induced endothelial hyperpermeability in vitro and in vivo

“…The permeability of isolated venules was determined according to a previous study. 13 Animals were injected intraperitoneally with AGEs (10 mg/kg) 14 and/or the inhibitor of MLCK, ML-7 (1 mg/kg), 15 and anaesthetized with a combination of ketamine (75 mg/kg) and midazolam (2.5 mg/kg) 6 h later. A suitable mesentery (diameter 60-0 µm, length 1.0-1.2 mm, no branch) was excised, immediately transferred into a cold albumin-physiological salt solution (APSS) separate pool and then mounted to a Zeiss microscope.…”

Interferon-Stimulated Gene 15 Upregulation Precedes the Development of Blood–Brain Barrier Disruption and Cerebral Edema after Traumatic Brain Injury in Young Mice

“…9 EB dye (2%, 10 l/g, i.p. Alpha Aesar, Fisher Scientific) was injected to PND21 and PND24 (impact depth 2 and 2.25mm) and sham mice (n=6 in each group) at 4h prior to sacrifice at 24h, 48h, 72h and 7 days following impact.…”

“…9 Mice were sacrificed with CO 2 and whole brains were removed, immediately weighed and then placed at 60 o C for 3 days. Dried whole brains were then re-weighed and the ratio of wet/dry was calculated, as we described previously.…”

“…9 Hang times (latency to fall in seconds (s)) for each treatment group were averaged. In brief, mice were individually placed on a wire cage lid until footing was established.…”

“…Scoring was as follows: nose touch = 1 point; two paws on object = 2 points; all four paws on top of object = 3 points, as we previously described. 9 Points were tallied and touches to old vs. new objects were compared. An observer blinded to the impact groups conducted video analysis.…”