Jee Y. Lee

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

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.

Valproic acid attenuates blood–spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury

J. Neurochem. (2012) 121, 818–829.

Acupuncture-mediated inhibition of inflammation facilitates significant functional recovery after spinal cord injury.

Here, we first demonstrated the neuroprotective effect of acupuncture after SCI. Acupuncture applied at two specific acupoints, Shuigou (GV26) and Yanglingquan (GB34) significantly alleviated apoptotic cell death of neurons and oligodendrocytes, thereby leading to improved functional recovery after SCI. Acupuncture also inhibited caspase-3 activation and reduced the size of lesion cavity and extent of loss of axons. We also found that the activation of both p38 mitogen-activated protein kinase and resident microglia after injury are significantly attenuated by acupuncture. In addition, acupuncture significantly reduced the expression or activation of pro-nerve growth factor, proinflammatory factors such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, nitric oxide synthase, cycloxygenase-2, and matrix metalloprotease-9 after SCI. Thus, our results suggest that the neuroprotection by acupuncture may be partly mediated via inhibition of inflammation and microglial activation after SCI and acupuncture can be used as a potential therapeutic tool for treating acute spinal injury in human.

Inhibition of ROS-induced p38MAPK and ERK activation in microglia by acupuncture relieves neuropathic pain after spinal cord injury in rats

Acupuncture (AP) is currently used worldwide to relieve pain. However, little is known about its mechanisms of action. We found that after spinal cord injury (SCI), AP inhibited the production of superoxide anion (O(2)·), which acted as a modulator for microglial activation, and the analgesic effect of AP was attributed to its anti-microglial activating action. Direct injection of a ROS scavenger inhibited SCI-induced NP. After contusion injury which induces the below-level neuropathic pain (NP), Shuigou and Yanglingquan acupoints were applied. AP relieved mechanical allodynia and thermal hyperalgesia, while vehicle and simulated AP did not. AP also decreased the proportion of activated microglia, and inhibited both p38MAPK and ERK activation in microglia at the L4-5. Also, the level of prostaglandin E(2) (PGE2), which is produced via ERK signaling and mediates the below-level pain through PGE2 receptor, was reduced by AP. Injection of p38MAPK or ERK inhibitors attenuated NP and decreased PGE2 production. Furthermore, ROS produced after injury-induced p38MAPK and ERK activation in microglia, and mediated mechanical allodynia and thermal hyperalgesia, which were inhibited by AP or a ROS scavenger. AP also inhibited the expression of inflammatory mediators. Therefore, our results suggest that the analgesic effect of AP may be partly mediated by inhibiting ROS-induced microglial activation and inflammatory responses after SCI and provide the possibility that AP can be used effectively as a non-pharmacological intervention for SCI-induced chronic NP in patients.

Neuroprotective effect of Scutellaria baicalensis on spinal cord injury in rats

Inflammation has been known to play an important role in the pathogenesis after spinal cord injury (SCI). Microglia are activated after injury and produce a variety of proinflammatory factors such as tumor necrosis factor‐α, interleukin‐1β, cyclooxygenase‐2, and reactive oxygen species leading to apoptosis of neurons and oligodendrocytes. In this study, we examined the neuroprotective effects of total ethanol extract of Scutellaria baicalensis (EESB), after SCI. Using primary microglial cultures, EESB treatment significantly inhibited lipopolysaccharide‐induced expression of such inflammatory mediators as tumor necrosis factor‐α, IL‐1β, IL‐6, cyclooxygenase‐2, and inducible nitric oxide synthase. Furthermore, reactive oxygen species and nitric oxide production were significantly attenuated by EESB treatment. For in vivo study, rats that had received a moderate spinal cord contusion injury at T9 received EESB orally at a dose of 100 mg/kg. EESB inhibited expression of proinflammatory factors and protein carbonylation and nitration after SCI. EESB also inhibited microglial activation at 4 h after injury. Furthermore, EESB significantly inhibited apoptotic cell death of neurons and oligodendrocytes and improved functional recovery after SCI. Lesion cavity and myelin loss were also reduced following EESB treatment. Thus, our data suggest that EESB significantly improve functional recovery by inhibiting inflammation and oxidative stress after injury.

Ghrelin Inhibits Hydrogen Peroxide-Induced Apoptotic Cell Death of Oligodendrocytes Via ERK and p38MAPK Signaling

Here, we examined the protective effect of ghrelin on apoptotic cell death induced by hydrogen peroxide (H₂O₂) in primary oligodendrocyte cultures. Ghrelin receptor, growth hormone secretagogue receptor 1a, was expressed in mature oligodendrocytes. H₂O₂ (1 mm) treatment induced apoptotic cell death of oligodendrocytes, which was significantly inhibited by ghrelin treatment. Ghrelin also reduced cytochrome c release, and caspase-3 activation increased by H₂O₂ treatment. Furthermore, the protective effect of ghrelin against H₂O₂-induced oligodendrocyte cell death was mediated through growth hormone secretagogue receptor 1a. Both ERK and p38MAPK were activated (peaked at 8 h in ERK and 1 h in p38MAPK) by H₂O₂ treatment, whereas c-Jun N-terminal kinase and Akt were not. Interestingly, ghrelin further increased ERK activation and decreased p38MAPK activation after H₂O₂ treatment. Next, we tried to elucidate the role of ERK and p38MAPK activation in H₂O₂-induced apoptotic cell death of oligodendrocytes using pharmacological inhibitors. We found that the inhibition of apoptotic cell death of oligodendrocytes by ghrelin was abolished by ERK inhibitor, PD98059 (20 μM), whereas cell survival was increased by p38MAPK inhibitor, SB203580 (10 μM). These results thus indicate that ghrelin inhibits H₂O₂ -induced oligodendrocytes cell death in part by increasing ERK activation and decreasing p38MAPK activation, and ghrelin may represent a potential therapeutic agent for protecting oligodendrocytes in central nervous system injuries.

Analgesic effect of acupuncture is mediated via inhibition of JNK activation in astrocytes after spinal cord injury.

Acupuncture (AP) has been used worldwide to relieve pain. However, the mechanism of action of AP is poorly understood. Here, we found that AP relieved neuropathic pain (NP) by inhibiting Jun-N-terminal kinase (JNK) activation in astrocytes after spinal cord injury (SCI). After contusion injury which induces the below-level (L4-L5) NP, Shuigou (GV26) and Yanglingquan (GB34) acupoints were applied. At 31 d after injury, both mechanical allodynia and thermal hyperalgesia were significantly alleviated by AP applied at GV26 and GB34. Immunocytochemistry revealed that JNK activation was mainly observed in astrocytes after injury. AP inhibited JNK activation in astrocytes at L4-L5 level of spinal cord. The level of p-c-Jun known, a downstream molecule of JNK, was also decreased by AP. In addition, SCI-induced GFAP expression, a marker for astrocytes, was decreased by AP as compared to control groups. Especially, the number of hypertrophic, activated astrocytes in laminae I–II of dorsal horn at L4-5 was markedly decreased by AP treatment when compared with vehicle and simulated AP-treated groups. When animals treated with SP600125, a specific JNK inhibitor, after SCI, both mechanical allodynia and thermal hyperalgesia were significantly attenuated by the inhibitor, suggesting that JNK activation is likely involved in SCI-induced NP. Also, the expression of chemokines which is known to be mediated through JNK pathway was significantly decreased by AP and SP600125 treatment. Therefore, our results indicate that analgesic effect of AP is mediated in part by inhibiting JNK activation in astrocytes after SCI.

Ghrelin inhibits BSCB disruption/hemorrhage by attenuating MMP-9 and SUR1/TrpM4 expression and activation after spinal cord injury.

Blood spinal cord barrier (BSCB) disruption after spinal cord injury (SCI) leads to secondary injury and results in apoptosis of neurons and glia, leading to permanent neurological deficits. Here, we examined the effect of ghrelin on BSCB breakdown and hemorrhage after SCI. After moderate weight-drop contusion injury at T9 spinal cord, ghrelin (80μg/kg) was administered via intraperitoneal injection immediately after SCI and then the same dose of ghrelin was treated every 6h for 1d. Our data showed that ghrelin treatment significantly inhibited the expression and activation of matrix metalloprotease-9 (MMP-9) at 1d after SCI. The increases of sulfonylurea receptor 1 (SUR1) and transient receptor potential melastatin 4 (TrpM4) expressions at 1h and 8h after SCI respectively were also alleviated by ghrelin treatment. In addition, both BSCB breakdown and hemorrhage at 1d after injury were significantly attenuated by ghrelin. In parallel, the infiltration of blood cells such as neutrophils and macrophages was inhibited by ghrelin treatment at 1d and 5d after SCI respectively. We also found that ghrelin receptor, growth hormone secretagogue receptor-1a (GHS-R1a), was expressed in the blood vessel of normal spinal tissue. Furthermore, the inhibitory effects of ghrelin on hemorrhage and BSCB disruption at 1d after SCI were blocked by GHS-R1a antagonist, [D-Lys-3]-GHRP-6 (3mg/kg). Thus, these results indicate that the neuroprotective effect by ghrelin after SCI is mediated in part by blocking BSCB disruption and hemorrhage through the down-regulation of SUR1/TrpM4 and MMP-9, which is dependent on GHS-R1a.

17β-Estradiol Inhibits MMP-9 and SUR1/TrpM4 Expression and Activation and Thereby Attenuates BSCB Disruption/Hemorrhage After Spinal Cord Injury in Male Rats

Blood-spinal cord barrier (BSCB) disruption and progressive hemorrhage after spinal cord injury (SCI) lead to secondary injury and the subsequent apoptosis and/or necrosis of neuron and glia, causing permanent neurological deficits. In this study, we examined the effect of 17β-estradiol (E2) on BSCB breakdown and hemorrhage as well as subsequent inflammation after SCI. After a moderate contusion injury at the 9th thoracic segment of spinal cord, E2 (300 μg/kg) was administered by iv injection immediately after SCI, and the same dose of E2 was then administered 6 and 24 hours after injury. Our data show that E2 attenuated BSCB permeability and hemorrhage and reduced the infiltration of neutrophils and macorphages after SCI. Consistent with this finding, the expression of inflammatory mediators was significantly reduced by E2. Furthermore, E2 treatment significantly inhibited the expression of sulfonylurea receptor 1 and transient receptor potential melastatin 4 after injury, which are known to mediate hemorrhage at an early stage after SCI. Moreover, the expression and activation of matrix metalloprotease-9 after injury, which is known to disrupt BSCB, and the degradation of tight junction proteins, such as zona occludens-1 and occludin, were significantly inhibited by E2 treatment. Furthermore, the protective effects of E2 on BSCB disruption and functional improvement were abolished by an estrogen receptor antagonist, ICI 182780 (3 mg/kg). Thus, our study provides evidence that the neuroprotective effect of E2 after SCI is, in part, mediated by inhibiting BSCB disruption and hemorrhage through the down-regulation of sulfonylurea receptor 1/transient receptor potential melastatin 4 and matrix metalloprotease-9, which is dependent on estrogen receptor.

An increase in voltage-gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury.

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage‐gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Nav1.1, Nav1.2, and Nav1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS‐induced NF‐κB activation, expression of TNF‐α, IL‐1β and inducible nitric oxide synthase, and NO production. LPS‐induced p38MAPK activation followed pro‐nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS‐induced JNK activation was not. TTX also inhibited caspase‐3 activation and cell death of primary cortical neurons in neuron/microglia co‐cultures by inhibiting LPS‐induced microglia activation. Furthermore, TTX attenuated caspase‐3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury. GLIA 2013;61:1807–1821