Thus, the reaction of the immune system induces a feed-forward response that produces a persistent pro-inflammatory microenvironment, which contributes to cell death and glial scar formation [ 2 , 7 ]. In addition, the persistent release of these pro-inflammatory modulators plays a critical role in the induction and maintenance of neuropathic pain [ 11 ]. Bromodomain and extra-terminal domain BET proteins belong to the reader family. BET proteins are characterized by the presence of two tandem bromodomains BD1 and BD2 and an extra-terminal domain ET , which are critical for their function [ 13 , 14 ].
These proteins bind to acetylated lysine residues in both histone and non-histone proteins to recruit transcriptional complexes and thus regulate gene expression. As a result, genes related to proliferation and inflammation are expressed [ 13 , 14 , 15 ]. Taking together, the inhibition of this epigenetic phenomenon is a promising target to control the inflammatory process. Given the importance of BET proteins in inflammation, we hypothesized that these epigenetic mediators might be a therapeutic target after SCI. We observed that JQ1 produced a decrease of pro-inflammatory and an increase of anti-inflammatory cytokine expression acutely after SCI.
We found that prolonged treatment with JQ1 reduced inflammatory response, increased neuroprotection, and enhanced functional outcome after SCI. General anesthesia was induced by an intraperitoneal injection i. Animals received a i. Vehicle-treated animals received only the vehicle solution. Sham mice underwent the same surgery except the contusion injury. Bone marrow-derived macrophages BMDMs were obtained from mouse femurs following a specific isolating protocol [ 17 ].
After the differentiation phase, adherent BMDMs were seeded at a density of 0. At days 9 and 10, DMSO 0. Finally, cells were collected for RNA extraction. Then, a 4-mm-long segment around the SCI epicenter was removed and snap-frozen.
This scale ranges from 0, indicating complete paralysis, to 9, indicating normal movement of the hind limbs. The analysis was carried out by two researchers which strictly followed blinding procedures to determine the score for each animal. To evaluate neuropathic pain, mechanical and thermal algesimetry tests were performed. Briefly, an increasing mechanical force was applied with a probe on the plantar surface of the hind paws until the animal produced retraction of the paw, indicating painful perception. Thermal algesimetry test [ 20 ] assesses the time that mice can endure heat irradiation.
Then, the area of residual white matter was expressed as the percentage with respect to the total area of the spinal cord section. The detection was made with appropriate secondary antibodies conjugated with Alexa Fluor A for Goat and A for Rabbit; Invitrogen.
The ROI had an area of 0. Finally, neuronal survival was assessed by counting the number of neurons located rostrally and caudally from the epicenter in both ventral horns. Measurements were performed with ImageJ software. For cytokine protein expression, unpaired t test was performed. After SCI, a large number of pro-inflammatory markers become upregulated. To investigate the efficacy of BET inhibition to modulate the secondary injury response, we monitored the expression of inflammation-related genes after SCI or in sham mice treated with the BET inhibitor JQ1 or with vehicle.
As shown in Fig. These results validate previous studies [ 21 ]. BET inhibitor JQ1 reduces expression of pro-inflammatory and increases expression of anti-inflammatory cytokines and chemokines acutely after SCI. In addition, our results revealed that the anti-inflammatory effect of JQ1 resides not only on reducing pro-inflammatory markers but also to upregulate anti-inflammatory cytokines. IL-4 levels were undetectable in most of the samples because the detection levels of the assay were too high for the biological levels of the protein data not shown.
Why Don't We Have a Cure for Spinal Cord Injury?
However, we corroborated the increase of IL and IL in sham and injured mice treated with JQ1 compared to vehicle-treated mice Fig. The protein levels of the pro-inflammatory cytokine IL-6 a and the anti-inflammatory cytokines IL and IL b were quantified via Luminex analysis at 4 and 72 h after operation.
In addition, other inflammatory markers related to macrophage infiltration and microglial activation were studied. Finally, we investigated whether the effects observed after BET inhibition in SCI could be produced by affecting macrophage reactivity. Overall, while JQ1 suppressed the expression of key pro-inflammatory genes, it also produced an early expression of anti-inflammatory cytokines after SCI. To further study the role of JQ1 in modulating the inflammatory response after SCI, we evaluated the expression of two hallmark markers of inflammation at longer time periods.
Iba1 staining detects both quiescent and reactive microglia and also infiltrated macrophages. Therefore, these results confirm that BET inhibition reduces microglial reactivity without affecting astroglial reactivity. JQ1 treatment reduces macrophage and microglia reactivity after SCI.
Besides, the total labeled myelin area was significantly higher in long-term JQ1-treated mice than in vehicle-treated mice Fig. While quantitative analysis of NeuN staining showed that most neurons died at the injury site and surrounding areas in the three groups of mice, neuron survival was significantly increased at different distances from the epicenter.
As it is shown in Fig. Overall, histopathological analysis revealed that long-term treatment of JQ1 conferred significant neuroprotection after SCI. Finally, neuropathic pain was evaluated with Randall-Selitto and thermal algesimetry tests Fig. The thermal algesimetry test showed a non-significant increase in the withdrawal threshold to hot stimulation in JQ1-treated mice. Although the interest in epigenetics has rapidly increased over the last years, the function of epigenetic signaling in central nervous system trauma remains largely unexplored.
Thus, the overall goal of this study was to investigate the therapeutic role of targeting the epigenome, specifically BET proteins, after SCI. For this purpose, we used in vitro and in vivo approaches to study the effect of the BET inhibitor JQ1 on inflammation, neuroprotection, functional recovery, and ultimately on neuropathic pain.
The results of the present study indicate that inhibition of BET proteins reduces inflammation and may represent a novel therapeutic target for SCI. We have observed that JQ1 decreased pro-inflammatory and increased anti-inflammatory cytokine expression acutely after SCI. We used JQ1 treatment at two different time regimes and observed that the prolonged treatment with JQ1 reduced the inflammatory response and increased neuroprotection.
Altogether, these neuroimmune outcomes were associated with an improvement of functional recovery and reduction in neuropathic pain. Overall, the results of the present study highlight the importance of epigenetic regulation on inflammation after SCI. One of the major findings of the present study is that BET inhibition, besides reducing pro-inflammatory cytokine expression, which had been previously described [ 15 , 21 ], enhances anti-inflammatory cytokine production.
Neuroprotection and regeneration in the spinal cord via the modulation of the prostaglandin pathway
This effect was observed after SCI and in a primary culture of bone marrow-derived macrophages. BET proteins are epigenetic readers, i. Thus, its inhibition commonly reduces gene expression.
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The reduction of these inflammatory-related genes, which are mediated by NF-kB signaling, can be explained by the disruption of the normal functioning of BET proteins by JQ1 treatment during an inflammatory response [ 22 , 23 ]. However, molecular mechanisms for increasing anti-inflammatory cytokine production after BET inhibition are elusive. This effect may be indirect. However, BET protein BRD4 can confer transcriptional repression by interacting with several repressive complexes [ 24 , 25 , 26 ].
Thus, it might be possible that BET inhibition treatment produces chromatin release of these complexes and triggers promoter de-repression as suggested previously [ 25 ], inducing increases of anti-inflammatory cytokine production. However, further experiments should be performed to decipher the molecular events undergoing these effects.
The importance of macrophages and microglia in SCI lies in their influence to exacerbate inflammation as well as repair. The raised levels of the anti-inflammatory cytokines IL-4, IL, and IL observed after JQ1 treatment are particularly interesting since many studies have highlighted their relevance in inducing an alternative activation to macrophages, which is linked to SCI recovery [ 1 , 27 , 28 ].
However, we found that the enhancement in anti-inflammatory cytokines could reduce the expression of the M1 marker iNOS but could not increase M2 markers ARG1 and CD , and thus, it might not affect the switch of macrophages into an anti-inflammatory phenotype. Accordingly to literature, this result could be explained because not all of the M1 and M2 markers seem to change expression in a coordinated fashion after SCI [ 5 ].mfo.mlsit.ru/libraries/y-reshebnik-po-4.php
Neuroprotection and Regeneration of the Spinal Cord | SpringerLink
Besides, it might be possible also that the dosage or timing of the treatment was not sufficient to induce a substantial increase of IL-4 to observe these effects. In fact, we were not able to detect IL-4 protein with the cytokine protein expression assay data not shown. We cannot affirm that BET inhibition did not enhance IL-4 production, since the detection range of the assay was higher than the protein biological levels. However, these levels are below those needed to produce a shift towards an M2 phenotype. Buy eBook. Buy Hardcover. Buy Softcover. Rent the eBook.
About this book Neuroprotection and Regeneration of the Spinal Cord comprehensively covers the most recent research in the field of spinal cord injury. Show all. B Micrographs of rat neurons cultured in control medium and FK with different doses on day 3. C Quantification of total neurite length following treatment with FK on days 1 and 3. Neurons were stained with microtubule associated protein 2 antibody red. A minimum of 80 neurons per condition were analyzed. Statistical comparisons between means were made with one-way analysis of variance.
The results showed that the total neurite length of individual neurons cultured with EGF was markedly longer compared with the control group Fig. EGF can promote neurite outgrowth. Total neurite length of spinal cord neurons cultured with EGF were markedly longer than the control group. To investigate the role of astrocytes as mediators of the neuroprotective effects of FK, the total and longest neurite length of spinal cord neurons cultivated with various CM for 1 and 3 days were measured. The results indicated that treatment with FKCM induced a After incubation with CMs for one day, the mean length of the total neurite of individual neurons cultured in FKCM was By contrast, the mean lengths of total neurites on day 3 were Similar results were observed when only the longest neurite was measured Figs.
Thus, analysis of neuronal morphology revealed a marked increase in the neurite outgrowth when the spinal neurons were treated with FKCM. FK was found to enhance neurite outgrowth on day 1 after treatments with CM. F Quantification of total neurite length and G longest neurite length of spinal cord neurons. The results indicated that FK was dependent on EGF stimulation in order to affect neurite outgrowth.
A minimum of 80 neurons in each of the CMs were analyzed. CM, conditioned media; C-CM, conditioned media of the control group. FK was found to enhance neurite outgrowth on the third day after treatment with CM. In the present study, gene chip detection of astrocytes treated with FK was performed. Furthermore, EGF displayed elevated levels of the cytokines, as shown in Fig.
Microarray results depicting the significantly elevated levels of cytokines subsequent to treatment with FK Following a contusion of the spinal cord, rats were randomly and blindly assigned to the FK 0. The results indicated that sections from FKtreated groups showed strongly EGF-immunoreactive astrocytes. These markedly EGF-immunoreactive astrocytes were predominantly in the vicinity of the lesion 24 h post-injury, reaching peak levels in the initial 3 days, and gradually decreasing until day 14 Fig.
By contrast, the EGF expression levels in the control group were markedly lower compared with those in the FKtreated group Fig. Additionally, the present study found that the level of EGF-immunoreactive astrocytes in the control group was perceptibly decreased on the third day compared with those detected at 24 h. After 7 days, few EGF-positive astrocytes remained in the control group. FK modulated astrocyte production of EGF in vivo. The expression levels of EGF in astrocytes was markedly increased in rats treated with FK on days 1, 3 and 7, compared with those in the normal control.
However, EGF expression was decreased markedly after day 7 post-surgery. To further verify the involvement of astrocyte-derived EGF on neurite outgrowth, embryonic spinal neurons were cultured in neutralized FKCM and neutralized C-CM, which had been pre-treated with EGF neutralizing antibodies, and subsequently the length of neurites were analyzed Figs. The total length of neurites was reduced by For day 3 these values were The same results were found with respect to the longest neurite length.
Furthermore, to exclude non-specific inhibitory effects with the solution or preservatives involved in the antibody preparation, control experiments were performed, which showed that none of these agents altered neurite elongation data not shown. Thus, the results indicated that astrocytic EGF secretion in response to FK treatment serves a significant role in neurite elongation. In the present study it was found that that FK was able to enhance neurite outgrowth and improve the functional recovery of the spinal cord by stimulating astrocytes to secrete EGF.
This is an indirect effect of FK on neuronal cells by an astrocyte-mediated process. Numerous studies support the hypothesis that FK is able to improve functional recovery and nerve regeneration after nerve injury 23 , In accordance with previous studies, the present results showed that FK could improve locomotor functional recovery of the limbs of rats after SCI, as quantified by the BBB score. In addition, a bell-shaped dose-response curve of FK on the rate of axonal regeneration and on neurite outgrowth has been reported 26 , Reactive astrogliosis is initiated when trigger factors produced at the site of injury drive astrocytes to become activated from their quiescent state The reactive astrocytes are reported to have a dual role with respect to their overall beneficial or detrimental effect on neuroprotection, tissue regeneration and functional recovery A study by Bush et al 29 demonstrated that ablation of proliferating astrocytes after SCI could lead to increased neuronal degeneration and motor deficits.
Therefore, astrocytes responses at the site of injury may contribute to neuroprotection and functional recovery after SCI. Additionally, Szydlowska et al 30 demonstrated that FK could block the activation of extracellular signal-regulated kinases 1 and 2 signaling in glutamate-induced death of astrocytes and astrocytic cell death in vitro and in ischemic brains. We hypothesized that FK could provide a neuroprotective effect by modulating the activity of astrocytes.
Szydlowska et al 31 reported that FK may inhibit glutamate-induced astrocyte death. In the present study, spinal neuronal cells were cultured with FKCM, which was the supernatant of FKtreated astrocyte culture, and a marked increase in neurite length was observed.
Thus, the results suggested that FK may stimulate astrocytes to secrete certain cytokines, which could significantly enhance neurite outgrowth. EGF is a highly mitogenic factor in numerous mammalian cell types 33 , and is able to promote the proliferation and differentiation of neuronal progenitors, postmitotic neurons and glial cells in the central nervous system 34 , EGF is also an important neurotrophic factor and can stimulate neurite outgrowth in a previous study In addition, studies have reported that EGF can modulate neurite extension by stimulating thyroid hormones and versican G3 domain 37 , Modulation of the expression of glial-derived neurotrophic factor has been considered as a potential neuroprotective mechanism of immunophilin ligands Furthermore, the results in the current study showed that astrocytes could be stimulated to secrete EGF by treatment with FK, which is a potent neurotrophic factor and could enhance neurite outgrowth in neuronal cell lines.
Thus, we suggest that the astrocytic EGF secretion in response to FK treatment serves an important role in the neuroprotective effect of FK However, the current study only reported the effects of the administration of FK within 30 min post-SCI at a single dose. Further experiments and studies are required to examine the consequence of repeated treatment with different doses of FK Furthermore, the present study was not able to elucidate the concrete mechanism underlying EGF-induced neurite outgrowth, and further efforts are warranted to clarify this mechanism in future investigations.
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