P-glycoprotein Expression Is Upregulated in a Pre-Clinical Model of Traumatic Brain Injury

Abstract

Athletes participating in contact sports are at risk for sustaining repeat mild traumatic brain injury (rmTBI). Unfortunately, no pharmacological treatment to lessen the pathophysiology of brain injury has received U.S. Food and Drug Administration (FDA) approval. One hurdle to overcome for potential candidate agents to reach effective therapeutic concentrations in the brain is the blood-brain barrier (BBB). Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp), line the luminal membrane of the brain capillary endothelium facing the vascular space. Although these transporters serve to protect the central nervous system (CNS) from damage by effluxing neurotoxicants before they can reach the brain, they may also limit the accumulation of therapeutic drugs in the brain parenchyma. Thus, increased Pgp expression following brain injury may result in reduced brain availability of therapeutic agents. We therefore questioned if repeat concussive injury increases Pgp expression in the brain. To answer this question, we used a rodent model of repeat mild closed head injury (rmCHI) and examined the messenger RNA (mRN) and protein expression of both isoforms of rodent Pgp (Abcb1a and Abcb1b). Compared with sham-operated controls (n = 5), the mRNA levels of both Abcb1a and Abcb1b were found to be increased in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury. Using a validated antibody, we show increased immunolabeling for Pgp in the dorsal cortex at day 5 and in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury compared with sham controls (n = 6). Taken together, these results suggest that increased expression of Pgp after rmCHI may reduce the brain accumulation of therapeutic drugs that are Pgp substrates. It is plausible that including a Pgp inhibitor with a candidate therapeutic agent may be an effective approach to treat the pathophysiology of rmCHI.

Keywords: P-glycoprotein; blood–brain barrier; hippocampus; traumatic brain injury.

FIG. 1.

rmCHI triggers an inflammatory state but not overt neuronal cell loss. Representative images of brain sections from (A) sham and (B) rmCHI animals 24 h after the last injury or sham surgery, stained with the neuronal marker NeuN (green), the astrocyte maker GFAP (red), and the microglial marker Iba-1 (blue). The boxed areas in (A) indicate the regions of the cortex and hippocampus that are shown in the higher magnification panels. Dashed lines indicate the position of the CA1/3 and dentate granule cell body layers in the hippocampus. Scale bars: whole brain panel: 1 mm; hippocampus panels: 200 μm; cortex panels: 100 μm. GFAP, glial fibrillary acidic protein; rmCHI, repeat mild closed head injury.

FIG. 2.

RT-PCR of ABC transport proteins in Sham, 1 day post-rmCHI, and 5 days post-rmCHI. Expression shown as normalized to L32. (A) Abcb1a; (B) Abcb1b; (C) Abcg2; (D) Abcc1. Overall effect is shown. *denotes p < 0.05; #denotes p < 0.01. ABC, ATP-binding cassette; rmCHI, repeat mild closed head injury; RT-PCR, real-time polymerase chain reaction.

FIG. 3.

Validation of AbCam anti-Pgp antibody (#ab170904). (A) Antibody validation in wild-type animals via Wes. (B) Antibody validation in KO animals via Wes. (C) Antibody validation via confocal imaging in wild-type and KO animals. Overall effect is shown. *denotes p < 0.05; #denotes p < 0.01. KO, knockout; Pgp, P-glycoprotein.

FIG. 4.

Immunolabeling of P-glycoprotein (Pgp) in sham, 1 day post-rmCHI, and 5 days post-rmCHI. (A) Dorsal cortex; (B) hippocampus. Overall effect is shown. *denotes p < 0.05; #denotes p < 0.01. Pgp, P-glycoprotein; rmCHI, repeat mild closed head injury.

FIG. 5.

RT-PCR of BBB proteins in sham, 1 day post-rmCHI, and 5 days post-rmCHI. (A) Cldn1; (B) Cldn5; (C) Ocln; (D) Tjp1. Overall effect is shown. *denotes p < 0.05; #denotes p < 0.01. BBB, blood–brain barrier; Cldn1, claudin 1; Cldn5, claudin 5; rmCHI, repeat mild closed head injury; RT-PCR, real-time polymerase chain reaction; Tjp1, zona occludens.