Fluoxetine inhibits matrix metalloprotease activation and prevents disruption of blood–spinal cord barrier after spinal cord injury

Abstract

After spinal cord injury, the disruption of blood-spinal cord barrier by activation of matrix metalloprotease is a critical event leading to infiltration of blood cells, inflammatory responses and neuronal cell death, contributing to permanent neurological disability. Recent evidence indicates that fluoxetine, an anti-depressant drug, is shown to have neuroprotective effects in ischaemic brain injury, but the precise mechanism underlying its protective effects is largely unknown. Here, we show that fluoxetine prevented blood-spinal cord barrier disruption via inhibition of matrix metalloprotease activation after spinal cord injury. After a moderate contusion injury at the T9 level of spinal cord with an infinite horizon impactor in the mouse, fluoxetine (10 mg/kg) was injected intraperitoneally and further administered once a day for indicated time points. Fluoxetine treatment significantly inhibited messenger RNA expression of matrix metalloprotease 2, 9 and 12 after spinal cord injury. By zymography and fluorimetric enzyme activity assay, fluoxetine also significantly reduced matrix metalloprotease 2 and matrix metalloprotease 9 activities after injury. In addition, fluoxetine inhibited nuclear factor kappa B-dependent matrix metalloprotease 9 expression in bEnd.3, a brain endothelial cell line, after oxygen-glucose deprivation/reoxygenation. Fluoxetine also attenuated the loss of tight junction molecules such as zona occludens 1 and occludin after injury in vivo as well as in bEnd.3 cultures. By immunofluorescence staining, fluoxetine prevented the breakdown of the tight junction integrity in endothelial cells of blood vessel after injury. Furthermore, fluoxetine inhibited the messenger RNA expression of chemokines such as Groα, MIP1α and 1β, and prevented the infiltration of neutrophils and macrophages, and reduced the expression of inflammatory mediators after injury. Finally, fluoxetine attenuated apoptotic cell death and improved locomotor function after injury. Thus, our results indicate that fluoxetine improved functional recovery in part by inhibiting matrix metalloprotease activation and preventing blood-spinal cord barrier disruption after spinal cord injury. Furthermore, our study suggests that fluoxetine may represent a potential therapeutic agent for preserving blood-brain barrier integrity following ischaemic brain injury and spinal cord injury in humans.

Regulation of Caveolin-1 and Junction Proteins by bFGF Contributes to the Integrity of Blood–Spinal Cord Barrier and Functional Recovery

“…Figure 2(E) shows that MMP-9 activation was markedly enhanced at 1 d after injury, consistent with previous reports [29], with bands corresponding to the active form of MMP-9 and the inactive zymogen (pro-MMP-9). Although pro-MMP-2 appeared in the SCI group, there was no active MMP-2, which is consistent with the report of Lee et al reports [25]. bFGF significantly decreased the level of active MMP-9 and pro-MMP-2 compared with the vehicle controls at 1 d after injury.…”

“…Moreover, the upregulation of MMP-2 also contributes to the initial opening of the BBB by degrading the basal lamina leading to neuronal injury [42]. Several studies have reported that active MMP-2 appeared at 5 d after SCI [25]. Interestingly, our results showed no obvious activation of MMP-2 at 1 d after injury (Fig.…”

“…The integrity of BSCB was investigated with Evans Blue dye and fluorescein isothiocyanate (FITC)-dextran (MW 70 kDa; Sigma-Aldrich) extravasation, according to previous reports [24,25]. At 1 day after SCI, animals were injected with 4 ml/ kg 2% Evans Blue (Sigma-Aldrich) into the tail vein.…”

“…Changes in the expression and distribution of TJ and AJ proteins are closely related to the permeability of BSCB during SCI [38]. The levels of occludin, or zonula occludens 1 (ZO-1), are decreased at 1 or 3 d after SCI [25,39]. It has been reported that occludin, claudin-5, and ZO-1 are lost or degraded at 48 h postinjury, which aggravates the disruption of BSCB integrity [40].…”

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP‐9 expression but not direct TJ proteins expression regulation

“…And MMP‐9 is among the most important MMPs in TJ proteins degradation 10, 12. Lee's study found that blocking MMP‐9 secretion by fluoxetine could alleviate TJ molecular degradation and BSCB disruption 10.…”