Osteopontin Promotes Angiogenesis in the Spinal Cord and Exerts a Protective Role Against Motor Function Impairment and Neuropathic Pain After Spinal Cord Injury.

STUDY DESIGN: Basic science study using a hemisection spinal cord injury (SCI) model. OBJECTIVE: We sought to assess the effect of blocking osteopontin (OPN) upregulation on motor function recovery and pain behavior after SCI and to further investigate the possible downstream target of OPN in the injured spinal cord. SUMMARY OF BACKGROUND DATA: OPN is a noncollagenous extracellular matrix protein widely expressed across different tissues. Its expression substantially increases following SCI. A previous study suggested that this protein might contribute to locomotor function recovery after SCI. However, its neuroprotective potential was not fully explored, nor were the underlying mechanisms. MATERIALS AND METHODS: We constructed a SCI mouse model and analyzed the expression of OPN at different time points and the particular cell distribution in the injured spinal cord. Then, we blocked OPN upregulation with lentivirus-delivering siRNA targeting OPN specifically and examined its effect on motor function impairment and neuropathic pain after SCI. The underlying mechanisms were explored in the OPN-knockdown mice model and cultured vascular endothelial cells. RESULTS: The proteome study revealed that OPN was the most dramatically increased protein following SCI. OPN in the spinal cord was significantly increased three weeks after SCI. Suppressing OPN upregulation through siRNA exacerbated motor function impairment and neuropathic pain. In addition, SCI resulted in an increase in vascular endothelial growth factor (VEGF), AKT phosphorylation, and angiogenesis within the spinal cord, all of which were curbed by OPN reduction. Similarly, OPN knockdown suppressed VEGF expression, AKT phosphorylation, cell migration, invasion, and angiogenesis in cultured vascular endothelial cells. CONCLUSION: OPN demonstrates a protective influence against motor function impairment and neuropathic pain following SCI. This phenomenon may result from the proangiogenetic effect of OPN, possibly due to activation of the VEGF and/or AKT pathways.

LncRNA MRAK159688 facilitates morphine tolerance by promoting REST-mediated inhibition of mu opioid receptor in rats.

Morphine tolerance (MT) caused by the long-term use of morphine is a major medical problem. The molecular mechanism of morphine tolerance remains elusive. Here, we established a morphine tolerance model in rats and verified whether the long noncoding RNA (lncRNA) MRAK159688 is involved in morphine tolerance and its specific molecular mechanism. We show the significant upregulation of MRAK159688 expression in the spinal cord of morphine-tolerant rats. Overexpression of MRAK159688 by a lentivirus reduces the analgesic efficacy of morphine and induces pain behavior. Downregulation of MRAK159688 using a small interfering RNA (siRNA) attenuates the formation of morphine tolerance, partially reverses the development of morphine tolerance and alleviates morphine-induced hyperalgesia. MRAK159688 is located in the nucleus and cytoplasm of neurons, and it colocalizes with repressor element-1 silencing transcription factor (REST) in the nucleus. MRAK159688 potentiates the expression and function of REST, thereby inhibiting the expression of mu opioid receptor (MOR) and subsequently inducing morphine tolerance. Moreover, REST overexpression blocks the effects of MRAK159688 siRNA on relieving morphine tolerance. In general, chronic morphine administration-mediated upregulation of MRAK159688 in the spinal cord contributes to morphine tolerance and hyperalgesia by promoting REST-mediated inhibition of MOR. MRAK159688 downregulation may represent a novel RNA-based therapy for morphine tolerance.

PPAR γ Prevents Neuropathic Pain by Down-Regulating CX3CR1 and Attenuating M1 Activation of Microglia in the Spinal Cord of Rats Using a Sciatic Chronic Constriction Injury Model.

BACKGROUND: Previous studies have proved that peripheral nerve injury is involved in the pathogenesis of neuropathic pain (NP). The peripheral nerve injury primes spinal M1 microglia phenotype and produces pro-inflammatory cytokines, which are responsible for neurotoxic and neuronal hyper-excitable outcomes. Spinal peroxisome proliferator-activated receptor gamma (PPAR γ) has been shown to play an anti-inflammatory role in the development of NP. However, the role of PPAR γ in attenuating the pathological pathway of spinal microgliosis is still unknown. METHODS: Sprague-Dawley rats (male, aged 8-10 weeks) were randomly divided into three groups, i.e., a control group, a NP group, and a NP + lentivirus encoding PPAR γ (LV-PPAR γ) group. The sciatic chronic constriction injury (CCI) model was used to induce NP in rats. Pain behavior was assessed by monitoring the rat hind-paw withdrawal threshold to mechanical stimuli and withdrawal latency to radiant heat. The LV-PPAR γ was intrathecally infused 1 day before CCI. Western blot analysis and real-time qPCR were used to detect the microglia phenotypic molecules and CX3CR1 expression in the spinal cord. In vitro, BV-2 microglia cells were transfected with LV-PPAR γ and incubated with lipopolysaccharides (LPS), and the levels of M1 microglia phenotypic molecules and CX3CR1 in BV-2 microglia cells were assessed by western blot analysis, real-time qPCR, and enzyme-linked immunosorbent assay. RESULTS: Preoperative intrathecal infusion of LV-PPAR γ attenuated pain in rats 7 days post-CCI. The M1-microglia marker, CX3CR1, and pro-inflammatory signaling factors were increased in the spinal cord of CCI rats, while the preoperative intrathecal infusion of LV-PPAR γ attenuated these changes and increased the expression of IL-10. In vitro, the overexpression of PPAR γ in BV-2 cells reduced LPS-induced M1 microglia polarization and the levels of CX3CR1 and pro-inflammatory cytokines. CONCLUSION: Intrathecal infusion of LV-PPAR γ exerts a protective effect on the development of NP induced by CCI in rats. The overexpression of PPAR γ may produce both analgesic and anti-inflammatory effects due to inhibition of the M1 phenotype and CX3CR1 signaling pathway in spinal microglia.

Transcriptomic analysis of long noncoding RNAs and mRNAs expression profiles in the spinal cord of bone cancer pain rats.

Bone cancer pain (BCP) is one of the most common types of chronic cancer pain and its pathogenesis has not been fully understood. Long non-coding RNAs (lncRNAs) are new promising targets in the field of pain research, however, their involvements in BCP have not been reported. In the present study, we established the BCP model by implantation of Walker 256 carcinoma cells into rats' tibial medullary cavity and performed transcriptome sequencing of the ipsilateral lumbar spinal cord to explore changes in expression profiles of lncRNA and mRNA. We identified 1220 differently expressed mRNAs (DEmRNAs) (1171 up-regulated and 49 down-regulated) and 323 differently expressed lncRNAs (DElncRNAs) (246 up-regulated and 77 down-regulated) in BCP model, among which 10 DEmRNAs (5 up-regulated and 5 down-regulated) and 10 DElncRNAs (5 up-regulated and 5 down-regulated) were validated the expression by RT-qPCR. Then, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on the expression of DEmRNAs and DElncRNAs, showing that they were mainly enriched in inflammatory and immunologic processes/pathways. Finally, we constructed a co-expression network and a ceRNA network of DEmRNAs and DElncRNAs to exhibit a potential regulatory mechanism of DElncRNAs, directly regulating protein coding gene expression in cis or in trans and indirectly regulating protein coding gene expression by sponging miRNA. In conclusion, our study provided a landscape of dysregulated lncRNA and mRNA in spinal cord of bone cancer pain and detected novel potential targets for treatment in the future.

Minocycline Relieves Depressive-Like Behaviors in Rats With Bone Cancer Pain by Inhibiting Microglia Activation in Hippocampus.

BACKGROUND: Pain and depression are highly prevalent symptoms in cancer patients. They tend to occur simultaneously and affect each other and share biological pathways and neurotransmitters. In this study, we investigated the roles of microglia in the hippocampus in the comorbidity of bone cancer pain and depressive-like behaviors in an animal model of bone cancer pain. METHODS: Bone cancer pain was induced by injection of Walker 256 mammary gland carcinoma cells into the tibia of rats. The effects of intracerebroventricular administration of microglia inhibitor minocycline were examined. RESULTS: Carcinoma intratibia injection caused comorbidity of mechanical allodynia and depressive-like behaviors in rats and activation of microglia in the hippocampus. Both mechanical allodynia and depressive-like behaviors were attenuated by minocycline. Enzyme-linked immunosorbent assay analysis showed that the enhanced expressions of M1 microglia marker (CD 86) and the proinflammatory cytokines tumor necrosis factor-α and interleukin-1ß in the hippocampus of cancer-bearing rats were decreased by minocycline. On the other hand, minocycline also increased the expressions of M2 microglia marker (MRC1) and anti-inflammatory cytokine interleukin-10. CONCLUSIONS: The results suggest that the activation of microglia in the hippocampus plays an important role in the development of pain and depressive-like behaviors in bone cancer condition.

Circular RNA expression profile in the spinal cord of morphine tolerated rats and screen of putative key circRNAs.

Morphine tolerance developed after repeated or continuous morphine treatment is a global health concern hindering the control of chronic pain. In our previous research, we have reported that the expression of lncRNAs and microRNAs have been greatly modified in the spinal cord of morphine tolerated rats, and the modulating role of miR-873a-5p, miR-219-5p and miR-365 have already been confirmed. However, whether circular RNAs, another essential kind of non-coding RNA, are involved in the pathogenesis of morphine tolerance is still beyond our knowledge. In this study, we conducted microarray analysis for circRNA profile and found a large number of circRNAs changed greatly in the spinal cord by morphine treatment. Among them, we selected nine circRNAs for validation, and seven circRNAs are confirmed. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) analysis were used for functional annotation. Besides, we confirmed the modified expression of seven circRNAs after validation by real-time PCR, selected 3 most prominently modulated ones among them and predicted their downstream miRNA-mRNA network and analyzed their putative function via circRNA-miRNA-mRNA pathway. Finally, we enrolled the differentially expressed mRNAs derived from the identical spinal cord, these validated circRNAs and their putative miRNA targets for ceRNA analysis and screened a promising circRNA-miRNA-mRNA pathway in the development of morphine tolerance. This study, for the first time, provided valuable information on circRNA profile and gave clues for further study on the circRNA mechanism of morphine tolerance.

Risk factors and outcomes of urosepsis in patients with calculous pyonephrosis receiving surgical intervention: a single-center retrospective study.

BACKGROUND: Urosepsis is a catastrophic complication, which can easily develop into septic shock and lead to death if not diagnosed early and effectively treated in time. However, there is a lack of evidence on the risk factors and outcomes in calculous pyonephrosis patients. Therefore, this study was conducted to identify risk factors and outcomes of intra- and postoperative urosepsis in this particular population. METHODS: Clinical data of 287 patients with calculous pyonephrosis were collected. In the univariate and multivariate analysis, all patients were divided into urosepsis group and non-urosepsis group. The diagnosis of urosepsis was mainly on the basis of the criteria of American College of Chest Physicians (ACCP)/Society of Critical Care Medicine (SCCM). Patient characteristics and outcomes data were analyzed, and risk factors were assessed by binary logistic regression analysis. RESULTS: Of 287 patients, 41 (14.3%) acquired urosepsis. Univariate analysis showed that white blood cell (WBC > 10*10^9/L) before surgery (P = 0.027), surgery types (P = 0.009), hypotension during surgery (P < 0.001) and urgent surgery (P < 0.001) were associated with intra- and postoperative urosepsis for calculous pyonephrosis patients. In multivariate analysis, hypotension during surgery and urgent surgery were closely related to intra- and postoperative urosepsis. Outcome analysis suggested that patients developing urosepsis had a longer intensive care unit (ICU) stay and postoperative hospital stay and higher mortality. CONCLUSIONS: Hypotension during surgery and urgent surgery were risk factors of intra- and postoperative urosepsis for calculous pyonephrosis patients, which may lead to a prolonged ICU stay, postoperative hospital stay and higher mortality.

Normalizing HDAC2 Levels in the Spinal Cord Alleviates Thermal and Mechanical Hyperalgesia After Peripheral Nerve Injury and Promotes GAD65 and KCC2 Expression.

Neuropathic pain is a worldwide health concern with poor treatment outcomes. Accumulating evidence suggests that histone hypoacetylation is involved in development and maintenance of neuropathic pain. Thus, many natural and synthetic histone deacetylase (HDACs) inhibitors were tested and exhibited a remarkable analgesic effect against neuropathic pain in animals. However, studies evaluating specific subtypes of HDACs contributing to neuropathic pain are limited. In this study, using the chronic constriction injury (CCI) rat model, we found that mRNA and protein levels of HDAC2 were increased in the lumbar spinal cord of rats after sciatic nerve injury. Intrathecal injection of TSA, a pan-HDAC inhibitor, suppressed the increase in HDAC2 protein but not mRNA, and showed a dose-dependent pain-relieving effect. By introducing HDAC2-specific shRNA into the spinal cord via a lentivirus vector, we confirmed that HDAC2 mediates mechanical and thermal hyperalgesia after nerve injury. Further examination found two essential participants in neuropathic pain in the inhibitory circuit of the central nervous system: GAD65 and KCC2 were increased in the spinal cord of CCI rats after HDAC2 knockdown. Thus, our research confirmed that HDAC2 was involved in mechanical and thermal hyperalgesia induced by peripheral nerve injury. Furthermore, GAD65 and KCC2 were the possible downstream targets of HDAC2 in pain modulation pathways.

Neonatal Lipopolysaccharide Challenge Induces Long-lasting Spatial Cognitive Impairment and Dysregulation of Hippocampal Histone Acetylation in Mice.

Neonatal inflammation induces long-term effects on brain function. We investigated the effects of systematic neonatal inflammation using lipopolysaccharide (LPS) injection at postnatal day 3 (P3) and P5 in a mouse model of spatial memory capacity measured using a Morris water maze (MWM) task in adulthood. Subsequently, we assessed histone acetylation and immediate-early response gene expression (c-Fos and brain-derived neurotrophic factor) in the hippocampus in response to MWM acquisition training. The LPS-treated mice exhibited a significant spatial cognitive impairment, which was accompanied by insufficient histone acetylation of the H4K12-specific lysine residue and repressed c-Fos gene expression immediately after acquisition training. Moreover, the enrichment of acetyl-H4K12 on the c-Fos promoter following acquisition training was decreased in LPS-treated mice. Administration of trichostatin A (TSA), a histone deacetylase inhibitor, 2 h before each MWM acquisition training session effectively enhanced hippocampal histone acetylation levels and enrichment of acetyl-H4K12 on the c-Fos promoter following acquisition training in LPS-treated mice. TSA also increased c-Fos gene expression underlying synaptic plasticity and memory formation, and consequently rescued impaired spatial cognitive function. These results indicate that the dysregulation of H4K12 acetylation during the ongoing process of memory formation plays a key role in the spatial cognitive impairment associated with a neonatal LPS challenge. The histone deacetylase inhibitor TSA exhibits therapeutic potential for treating cognitive impairment induced by neonatal inflammation, by means of improving hippocampal histone acetylation and downstream c-Fos gene expression in response to a learning task.

[Trichostatin A suppresses up-regulation of histone deacetylase 4 and reverses differential expressions of miRNAs in the spinal cord of rats with chronic constrictive injury].

OBJECTIVE: To explore the analgesic mechanism of intrathecal trichostatin A (TSA) injection in a rat model of neuropathic pain induced by chronic constrictive injury (CCI). METHODS: Male SD rats were randomized into sham operation+ DMSO group (group S), CCI +DMSO group (group C), CCI +10 µg TSA group (group T), and in the latter two groups, rat models of neuropathic pain were established induced by CCI. The rats were given intrathecal injections of 10 µL 5% DMSO or 10 µg TSA (in 5% DMSO) once a day on days 7 to 9 after CCI or sham operation. The rats were euthanized after behavioral tests on day 10, and the lumbar segment of the spinal cord was sampled to determine the expression of histone deacetylase 4 (HDAC4) protein and mRNA and detect the differentially expressed miRNAs using a miRNA chip. MiR-190b-5p and miR-142-3p were selected for validation of the results using RT-qPCR. RESULTS: Compared with those in group S, the rats in group C showed significantly decreased paw withdrawal mechanical threshold (PWMT) from day 3 to day 10 after CCI (P < 0.05); intrathecal injection of TSA significantly reversed the reduction of PWMT following CCI (P < 0.05). Positive HDAC4 expression was detected mainly in the cytoplasm of the neurons in the gray matter of the spinal cord, and was obviously up-regulated after CCI (Ρ < 0.05). Intrathecal injection of TSA significantly suppressed CCI-induced up-regulation of HDAC4 at 10 days after the operation (P < 0.05). Compared with the miRNA profile in group S, miRNA profiling identified 83 differentially expressed miRNAs in group C (fold change ≥2 or ≤0.5, P < 0.05); TSA treatment reversed the expressions of 58 of the differentially expressed miRNAs following CCI, including 41 miRNAs that were decreased after CCI but up-regulated following TSA treatment. The results of real-time PCR validated the changes in the expressions of miR-190b-5p and miR-142-3p. CONCLUSIONS: TSA suppresses CCI-induced up-regulation of HDAC4 and reverses differential expressions of miRNAs in the spinal cord of rats, which may contribute to the analgesic effect of TSA on neuropathic pain.

Sufentanil Alleviates Intrathecal Lidocaine Induced Prolonged Sensory and Motor Impairments but not the Spinal Histological Injury in Rats.

Spinal anesthesia has evolved into a safe and widely accepted method of anesthesia. Synergy between opioids and local anesthetics further increases the quality of analgesia and decreases the dose requirement of both local anesthetics and opioids. However, over the last decades compelling evidence suggested that lidocaine could be more neurotoxic than other commonly used local anesthetics. Whether opioids can modify the local anesthetics-induced neurotoxicity is largely unexplored. Here, we investigated the effect of sufentanil on the neurotoxicity induced by intrathecal lidocaine in a rat model. Our data showed that 5 µg/ml sufentanil didn't deteriorate nor reduce the histopathological injuries induced by intrathecal application of 10% lidocaine in a rat model. However, it did alleviate sensory and motor function impairments induced by 10% lidocaine. Repeated intrathecal injection of 5 µg/ml sufentanil also decreased the paw withdraw threshold compared to the baseline. An increase in expression of activating transcription factor 3, a stress response gene, as a marker for injured neurons, was also detected in lidocaine-induced neurotoxicity, while 5 µg/ml sufentanil inhibited lidocaine-induced the upregulation of activating transcription factor 3. These results suggest that sufentanil alleviates lidocaine induced sensory and motor impairments, and did not worsen histopathological injury induced by intrathecal lidocaine.

Suppression of HDAC2 in Spinal Cord Alleviates Mechanical Hyperalgesia and Restores KCC2 Expression in a Rat Model of Bone Cancer Pain.

Epigenetic modulation participates in the mechanism of multiple types of pathological pain, so targeting the involved regulators may be a promising strategy for pain treatment. Our previous research identified the analgesic effect of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on mechanical hyperalgesia in a rat model of bone cancer pain (BCP) via restoration of µ-opioid receptor (MOR) expression. However, the specific types of HDACs contributing to BCP have not been explored. The present study investigated the expression pattern of some common HDACs and found that HDAC2 was up-regulated in a time-dependent manner in the lumbar spinal cord of BCP rats. TSA application suppressed HDAC2 expression in cultured PC12 cells and reversed the augmented HDAC2 in BCP rats. An RNA-interfering strategy confirmed the essential role of HDAC2 in the modulation of mechanical hyperalgesia following tumor cell inoculation, and we further examined its possible downstream targets. Notably, HDAC2 knock-down did not restore MOR expression, but it robustly reversed the down-regulation of potassium-chloride cotransporter 2 (KCC2). The impaired KCC2 expression is a vital mechanism of many types of pathological pain. Therefore, our results demonstrated that HDAC2 in spinal cord contributed to the mechanical hyperalgesia in BCP rats, and this effect may be associated with KCC2 modulation.

Proteomic Identification of an Upregulated Isoform of Annexin A3 in the Spinal Cords of Rats in a Neuropathic Pain Model.

Neuropathic pain (NP) is induced by nerve damage or a disturbance in the peripheral or central nervous systems. Nerve damage causes the activation of sensitizing mechanisms in the peripheral and central nervous systems, which induces transcriptional and post-transcriptional alterations in sensory nerves. However, the underlying mechanisms of NP remain elusive. In the study, Two-dimensional gel electrophoresis (2DGE)-based comparative proteomics identified 38 differential gel spots, and 15 differentially expressed proteins (DEPs) between the sham and the chronic constriction injury (CCI)-induced neuropathic pain rats. Of them, Annexin A3 (ANXA3) was significantly increased after CCI with Western blot analysis and immunofluorescence imaging. A lentivirus delivering ANXA3 shRNA (LV-shANXA3) was administered intrathecally to determine the analgesic effects of ANXA3 on allodynia and hyperalgesia in a CCI-induced neuropathic pain model in rats. Further study showed that LV-shANXA3 reversed the upregulation of ANXA3, alleviated CCI-induced mechanical allodynia and thermal hyperalgesia. The study indicated that ANXA3 may play an important role in neuropathic pain.

HDAC inhibitor TSA ameliorates mechanical hypersensitivity and potentiates analgesic effect of morphine in a rat model of bone cancer pain by restoring µ-opioid receptor in spinal cord.

Bone cancer pain (BCP) is a common complication with inadequate management in patients suffering from advanced cancer. Histone deacetylase inhibitors showed significant analgesic effect in multiple inflammatory and neuropathic pain models, but their effect in bone cancer pain has never been explored. In this study, we utilized a BCP rat model with intra-tibial inoculation of Walker 256 mammary gland carcinoma cells, which developed progressive mechanical hypersensitivity but not thermal hypersensitivity. Intrathecal application of trichostatin A (TSA), a classic pan-HDAC inhibitor, ameliorated tactile hypersensitivity and enhanced the analgesic effect of morphine in BCP rats. The analgesic effect of TSA was blocked by co-administration of CTAP, a specific MOR antagonist, confirming the involvement of mu-opioid receptor (MOR). A reduction of MOR expression was observed in the lumbar spinal cord of BCP rats and TSA treatment was able to partially reverse it. In vitro study in PC12 cells also demonstrated the dose-dependent enhancement of MOR expression by TSA treatment. Taking all into consideration, we could draw the conclusion that HDAC inhibitor TSA ameliorates mechanical hypersensitivity and potentiates analgesic effect of morphine in BCP rats, probably by restoring MOR expression in spinal cord.

Spatiotemporal changes in NSF expression of DRG neurons in a rat model of spinal nerve ligation.

N-ethylmaleimide-sensitive fusion (NSF) protein is a homohexameric ATPase that binds to the GluR2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptors. The stability and movement of AMPA receptors at synapses are important factors that control synaptic strength. NSF is involved in the surface expression regulation of AMPA receptors and consequently synaptic activity. Reduced expression of NSF or reduced interaction of NSF with GluR2 leads to a number of neurological disorders. Using a rat model of L5 spinal nerve ligation (SNL), we investigated the temporal and spatial expression of NSF in injured L5 and uninjured L4 dorsal root ganglion (DRG) neurons during mechanical allodynia. L5 SNL led to a significant decrease of NSF in both L4 and L5 DRGs observed at 3, 7, and 14 days after injury. In particular, NSF expression in calcitonin gene-related peptide (CGRP)-immunoreactive (IR) and IB4-IR neurons was reduced, whereas NSF expression in NF-200-IR neurons remained unaltered. These results indicate a role for NSF in CGRP-IR and IB4-IR neurons in SNL, with reduced NSF expression possibly contributing to SNL derived neuropathic pain.

Amyloid-ß plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound.

Noninvasive, targeted drug delivery to the brain can be achieved using transcranial focused ultrasound (FUS), which transiently increases the permeability of the blood-brain barrier (BBB) for localized delivery of therapeutics from the blood to the brain. Previously, we have demonstrated that FUS can deliver intravenously-administered antibodies to the brain of a mouse model of Alzheimer's disease (AD) and rapidly reduce plaques composed of amyloid-ß peptides (Aß). Here, we investigated two potential effects of transcranial FUS itself that could contribute to a reduction of plaque pathology, namely the delivery of endogenous antibodies to the brain and the activation of glial cells. We demonstrate that transcranial FUS application leads to a significant reduction in plaque burden four days after a single treatment in the TgCRND8 mouse model of AD and that endogenous antibodies are found bound to Aß plaques. Immunohistochemical and western blot analyses showed an increase in endogenous immunoglobulins within the FUS-targeted cortex. Subsequently, microglia and astrocytes in FUS-treated cortical regions show signs of activation through increases in protein expression and changes in glial size, without changes in glial cell numbers. Enhanced activation of glia correlated with increased internalization of Aß in microglia and astrocytes. Together these data demonstrate that FUS improved the bioavailability of endogenous antibodies and led to a temporal activation of glial cells, providing evidence towards antibody- and glia-dependent mechanisms of FUS-mediated plaque reduction.

Prostaglandin metabolite induces inhibition of TRPA1 and channel-dependent nociception.

BACKGROUND: The Transient Receptor Potential (TRP) ion channel TRPA1 is a key player in pain pathways. Irritant chemicals activate ion channel TRPA1 via covalent modification of N-terminal cysteines. We and others have shown that 15-Deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) similarly activates TRPA1 and causes channel-dependent nociception. Paradoxically, 15d-PGJ2 can also be anti-nociceptive in several pain models. Here we hypothesized that activation and subsequent desensitization of TRPA1 in dorsal root ganglion (DRG) neurons underlies the anti-nociceptive property of 15d-PGJ2. To investigate this, we utilized a battery of behavioral assays and intracellular Ca2+ imaging in DRG neurons to test if pre-treatment with 15d-PGJ2 inhibited TRPA1 to subsequent stimulation. RESULTS: Intraplantar pre-injection of 15d-PGJ2, in contrast to mustard oil (AITC), attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not capsaicin (CAP). Intraplantar 15d-PGJ2-administered after the induction of inflammation-reduced mechanical hypersensitivity in the Complete Freund's Adjuvant (CFA) model for up to 2 h post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity is dependent on TRPA1, as this effect was absent in TRPA1 knockout mice. Ca2+ imaging studies of DRG neurons demonstrated that 15d-PGJ2 pre-exposure reduced the magnitude and number of neuronal responses to AITC, but not CAP. AITC responses were not reduced when neurons were pre-exposed to 15d-PGJ2 combined with HC-030031 (TRPA1 antagonist), demonstrating that inhibitory effects of 15d-PGJ2 depend on TRPA1 activation. Single daily doses of 15d-PGJ2, administered during the course of 4 days in the CFA model, effectively reversed mechanical hypersensitivity without apparent tolerance or toxicity. CONCLUSIONS: Taken together, our data support the hypothesis that 15d-PGJ2 induces activation followed by persistent inhibition of TRPA1 channels in DRG sensory neurons in vitro and in vivo. Moreover, we demonstrate novel evidence that 15d-PGJ2 is analgesic in mouse models of pain via a TRPA1-dependent mechanism. Collectively, our studies support that TRPA1 agonists may be useful as pain therapeutics.

Early increases in soluble amyloid-ß levels coincide with cholinergic degeneration in 3xTg-AD mice.

Accumulation of amyloid-ß peptides (Aß) and cholinergic degeneration are hallmarks of Alzheimer's disease (AD). In a triple transgenic mouse model of AD (3xTg-AD), soluble Aß42 levels were detected in the septum by 2 months of age, reaching their highest levels at 3-6 months and decreasing at 12 months. Deficits in the number of septal cholinergic neurons and the length of hippocampal cholinergic axons were observed starting at 4 months in 3xTg-AD mice. Our results show that septal Aß and septohippocampal cholinergic pathology in 3xTg-AD mice occur at an early stage of disease.

Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound.

Noninvasive drug delivery to the brain remains a major challenge for the treatment of neurological disorders. Transcranial focused ultrasound combined with lipid-coated gas microspheres injected into the bloodstream has been shown to increase the permeability of the blood-brain barrier locally and transiently. Coupled with magnetic resonance imaging, ultrasound can be guided to allow therapeutics administered in the blood to reach brain regions of interest. Using this approach, we perform gene transfer from the blood to specific regions of the mouse brain. Focused ultrasound was targeted to the right hemisphere, at multiple foci, or restricted to one focal point of the hippocampus or the striatum. Doses from 5 × 10(8) to 1.25 × 10(10) vector genomes per gram (VG/g) of self-complementary adeno-associated virus serotype 9 carrying the green fluorescent protein were injected into the tail vein. A dose of 2.5 × 10(9) VG/g was optimal to express the transgene, 12 days later, in neurons, astrocytes, and oligodendrocytes in brain regions targeted with ultrasound, while minimizing the infection of peripheral organs. In the hippocampus and striatum, predominantly neurons and astrocytes were infected, respectively. Transcranial focused ultrasound applications could fulfill a long-term goal of gene therapy: delivering vectors to diseased brain areas directly from the circulation, in a noninvasive manner.