Changes in SLITRK1 Level in the Amygdala Mediate Chronic Neuropathic Pain-Induced Anxio-Depressive Behaviors in Mice.

BACKGROUND: Comorbid chronic neuropathic pain (NPP) and anxio-depressive disorders (ADD) have become a serious global public-health problem. The SLIT and NTRK-like 1 (SLITRK1) protein is important for synaptic remodeling and is highly expressed in the amygdala, an important brain region involved in various emotional behaviors. We examined whether SLITRK1 protein in the amygdala participates in NPP and comorbid ADD. METHODS: A chronic NPP mouse model was constructed by L5 spinal nerve ligation; changes in chronic pain and ADD-like behaviors were measured in behavioral tests. Changes in SLITRK1 protein and excitatory synaptic functional proteins in the amygdala were measured by immunofluorescence and Western blot. Adeno-associated virus was transfected into excitatory synaptic neurons in the amygdala to up-regulate the expression of SLITRK1. RESULTS: Chronic NPP-related ADD-like behavior was successfully produced in mice by L5 ligation. We found that chronic NPP and related ADD decreased amygdalar expression of SLITRK1 and proteins important for excitatory synaptic function, including Homer1, postsynaptic density protein 95 (PSD95), and synaptophysin. Virally-mediated SLITRK1 overexpression in the amygdala produced a significant easing of chronic NPP and ADD, and restored the expression levels of Homer1, PSD95, and synaptophysin. CONCLUSION: Our findings indicated that SLITRK1 in the amygdala plays an important role in chronic pain and related ADD, and may prove to be a potential therapeutic target for chronic NPP-ADD comorbidity.

Microglial voltage-dependent anion channel 1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

STUDY OBJECTIVES: This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial voltage-dependent anion channel 1 (VDAC1) signaling. METHODS: Adult mice received 6 hours of total sleep deprivation from 1 day prior to SMIR until the third day after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes in VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial adenosine 5' triphosphate (ATP) release, inflammation (IL-1ß and CCL2), and chronicity of pain. RESULTS: Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia, and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1ß and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. CONCLUSIONS: Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Combination of autophagy and NFE2L2/NRF2 activation as a treatment approach for neuropathic pain.

Macroautophagy/autophagy, an evolutionarily conserved process, plays an important role in the regulation of immune inflammation and nervous system homeostasis. However, the exact role and mechanism of autophagy in pain is still unclear. Here, we showed that impaired autophagy flux mainly occurred in astrocytes during the maintenance of neuropathic pain. No matter the stage of neuropathic pain induction or maintenance, activation of autophagy relieved the level of pain, whereas inhibition of autophagy aggravated pain. Moreover, the levels of neuroinflammation and reactive oxygen species (ROS) were increased or decreased following autophagy inhibition or activation. Further study showed that inhibition of autophagy slowed the induction, but increased the maintenance of neuroinflammatory responses, which could be achieved by promoting the binding of TRAF6 (TNF receptor-associated factor 6) to K63 ubiquitinated protein, and increasing the levels of p-MAPK8/JNK (mitogen-activated protein kinase 8) and nuclear factor of kappa light polypeptide gene enhancer in B cells (NFKB/NF-κB). Impaired autophagy also reduced the protective effect of astrocytes on neurons against ROS stress because of the decrease in the level of glutathione released by astrocytes, which could be improved by activating the NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2) pathway. We also demonstrated that simultaneous activation of autophagy and the NFE2L2 pathway further relieved pain, compared to activating autophagy alone. Our study provides an underlying mechanism by which autophagy participates in the regulation of neuropathic pain, and a combination of autophagy and NFE2L2 activation may be a new treatment approach for neuropathic pain.Abbreviation: 3-MA: 3-methyladenine; 8-OHdG: 8-hydroxydeoxy-guanosine; ACTB: actin, beta; AMPAR: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor; ATG: autophagy-related; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CCL7: chemokine (C-C motif) ligand 7; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; GABA: gamma-aminobutyrate; GCLC: glutamate-cysteine ligase, catalytic subunit; GFAP: glial fibrillary acidic protein; GSH: glutathione; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch-like ECH-associated protein 1; MAP1LC3/LC3-II: microtubule-associated protein 1 light chain 3 beta (phosphatidylethanolamine-conjugated form); MAPK: mitogen-activated protein kinase; MAPK1/ERK: mitogen-activated protein kinase 1; MMP2: matrix metallopeptidase 2; MAPK8/JNK: mitogen-activated protein kinase 8; MAPK14/p38: mitogen-activated protein kinase 14; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; ROS: reactive oxygen species; SLC12A5: solute carrier family 12, member 5; SNL: spinal nerve ligation; TLR4: toll-like receptor 4; TRAF6: TNF receptor-associated factor; TRP: transient receptor potential.

Amelioration of postoperative cognitive dysfunction in mice by mesenchymal stem cell-conditioned medium treatments is associated with reduced inflammation, oxidative stress and increased BDNF expression in brain tissues.

OBJECTIVE: Mesenchymal stem cells (MSCs) are widely used in regeneration and repair of various tissues and organs, and whether MSC-conditioned medium (MSC-CM) has protective effects in postoperative cognitive dysfunction (POCD) remains largely unknown. We aimed to assess the therapeutic effect and explore the mechanisms of MSC-CM therapy in a POCD mouse model. METHODS: Sixty C57BL/6 mice were randomly assigned to 3 groups: control, POCD and POCD + MSC-CM. The POCD mouse model was established by left liver lobectomy. While mice in the control group were sham-operated, mice in the POCD + MSC-CM group were immediately administrated with MSC-CM after operation. The Morris water maze was used to determine cognitive function of mice at 1, 3, and 7 days after operation. The levels of IL-1ß, IL-6, TNF-α and malondialdehyde in brain tissues at 3 days after operation were assessed by ELISA, while the protein level of brain derived neurotrophic factor (BDNF) was determined by western blot. RESULTS: Left liver lobectomy induced POCD in mice resulted in decrease of cognitive function, increase of brain IL-1ß, IL-6, TNF-α and malondialdehyde levels, and decreased BDNF expression, while administration of MSC-CM significantly reversed these changes. CONCLUSION: MSC-CM ameliorates POCD in mice, and its protective roles are associated with reduced levels of inflammatory factors, attenuated oxidative stress, and increased BDNF expression.

Fluoxetine suppresses inflammatory reaction in microglia under OGD/R challenge via modulation of NF-κB signaling.

We aimed to investigate the anti-inflammatory role of fluoxetine, a selective serotonin reuptake inhibitor, in microglia (MG) and the mechanisms under oxygen glucose deprivation/reoxygenation (OGD/R). An OGD/R model on BV-2 cells was used for the study of microglia under ischemia/reperfusion injury in ischemic stroke. Lentiviral transfection was applied to knock down IκB-α. Enzyme-linked immunosorbent assay (ELISA) was used for detecting levels of TNF-α, IL-1ß, and IL-6, and real-time PCR was used to assess the expression of IκB-α protein. Western blotting was applied to analyze NF-κB-signaling related proteins and Cell Counting Kit-8 (CCK-8) was used for assessing cell viability. Molecular docking and drug affinity responsive target stability (DARTS) assay were used for the detection of the interaction between IκB-α and fluoxetine. We found that fluoxetine decreased the levels of TNF-α, IL-1ß, and IL-6 in supernatant as well as NF-κB subunits p65 and p50 in BV-2 cells under OGD/R. Fluoxetine significantly increased the level of IκB-α through the inhibition of IκB-α ubiquitylation and promoted the bonding of IκB-α and fluoxetine in BV-2 cells under OGD/R. Knocking down IκB-α attenuated the decreasing effect of TNF-α, IL-1ß, and IL-6 as well as p65 and p50 in BV-2 cells under OGD/R led to by fluoxetine. In conclusion, our present study demonstrated the anti-inflammatory role of fluoxetine and its mechanisms related to the modulation of NF-κB-related signaling in MG under ischemia/reperfusion challenge.

Neuron-derived FGF10 ameliorates cerebral ischemia injury via inhibiting NF-κB-dependent neuroinflammation and activating PI3K/Akt survival signaling pathway in mice.

FGF10 is a member of fibroblast growth factors (FGFs). We previously showed that FGF10 protects neuron against oxygen-glucose deprivation injury in vitro; however, the effect of FGF10 in ischemic stroke in vivo is unknown. In the present study, we showed that FGF10 was mainly expressed in neurons but not astrocytes, and detected FGF10 in mouse cerebrospinal fluid. The FGF10 levels in neurons culture medium and cell lysate were much higher than those in astrocytes. FGF10 expression in brain tissue and FGF10 level in CSF were increased in mouse middle cerebral artery occlusion (MCAO) model. Administration of FGF10 into lateral cerebroventricle not only decreased MCAO-induced brain infarct volume and neurological deficit, but also reduced the number of TUNEL-positive cells and activities of Caspases. Moreover, FGF10 treatment depressed the triggered inflammatory factors (TNF-α and IL-6) and NF-κB signaling pathway, and increased phosphorylation of PI3K/Akt signaling pathway. Blockade of PI3K/Akt signaling pathway by wortmannin and Akt1/2-kinase inhibitor, partly compromised the neuroprotection of FGF10. However, blockade of PI3K/Akt signaling pathway did not impair the anti-inflammation action of FGF10. Collectively, our results demonstrate that neuron-derived FGF10 ameliorates cerebral ischemia injury via inhibiting NF-κB-dependent neuroinflammation and activating PI3K/Akt survival signaling pathway in mice.

Influence of Dexmedetomidine on the Tourniquet Related Responses in Hypertension Patients Receiving Unilateral Knee Arthroplasty under General Anesthesia.

This study aimed to investigate the influence of dexmedetomidine (DEX) on the tourniquet related responses in hypertension patients receiving unilateral knee arthroplasty (UKA) under general anesthesia. Results showed that the incidence of tourniquet induced hypertension (TIH), hemodynamics, MAC and EtSEV in DEX group were significantly lower than those in control group, regardless of hypertension. However, significant differences in TIH, hemodynamics, minimum alveolar concentration (MAC) and end-tidal sevoflurane (EtSEV) were not observed between hypertension patients and non-hypertension patients in both control group and DEX group. Moreover, oxygen index (OI) and respiratory index (RI) remained unchanged after deflation and DEX failed to affect OI and RI within 30 min after deflation, regardless of hypertension. Taken together, DEX may significantly improve the hemodynamics, which is independent of pre-existing hypertension.

(R)-alpha-methylhistamine suppresses inhibitory neurotransmission in hippocampal CA1 pyramidal neurons counteracting propofol-induced amnesia in rats.

BACKGROUND: Propofol is a short-acting, intravenous general anesthetic that is widely used in clinical practice for short procedures; however, it causes depressed cognitive function for several hours thereafter. (R)-alpha-methylhistamine (RAMH), a selective histamine H3 receptor agonist, can enhance memory retention and attenuates memory impairment in rats. In this study, we investigated whether RAMH could rescue propofol-induced memory deficits and the underlying mechanisms partaking in this process. METHODS: In the modified Morris water maze (MWM) test, rats were randomized into the following groups: control, propofol (25 mg/kg, i.p., 30 min before training), RAMH (10 mg/kg, i.p., 60 min before training), and propofol plus RAMH. All randomized rats were subjected to 2 days of training, and a probe test was conducted on day 3. Field excitatory postsynaptic potentials were recorded from CA1 neurons in rat hippocampal slices, and long-term potentiation (LTP) was induced by either theta-burst stimulation (TBS) or high-frequency tetanic stimulation (HFS). Spontaneous and miniature inhibitory (sIPSCs, mIPSCs) or excitatory (sEPSCs, mEPSCs) postsynaptic currents were recorded from CA1 pyramidal neurons by whole-cell patch clamp. RESULTS: In the MWM task, propofol injection significantly impaired spatial memory retention. Pretreatment with RAMH reversed propofol-induced memory retention. In hippocampal CA1 slices, propofol perfusion markedly inhibited TBS- but not HFS-induced LTP. Co-perfusion of RAMH reversed the inhibitory effect of propofol on TBS-induced LTP reduction. Furthermore, in hippocampal CA1 pyramidal neurons, RAMH significantly suppressed the frequency but not the amplitude of sIPSCs and mIPSCs and had little effects on both the frequency and amplitude of sEPSCs and mEPSCs. CONCLUSIONS: Our results suggest that RAMH, by inhibiting presynaptic GABAergic neurotransmission, suppresses inhibitory neurotransmission in hippocampal CA1 pyramidal neurons, which in turn reverses inhibition of CA1 LTP and the spatial memory deficits induced by propofol in rats.

Role of mast cell activation in inducing microglial cells to release neurotrophin.

The brain-derived neurotrophic factor (BDNF) plays a critical role in pain hypersensitivity. BDNF is the ligand of P2X4 receptors (P2X4R) in the microglia. The causative factors involving the P2X4R over expression in the microglia remains unclear. Mast cell activation has a close relation with pain hypersensitivity. However, the underlying mechanism between mast cell activation and pain hypersensitivity is unknown. The present study aimed to elucidate the mechanism by which mast cell activation promoted the expression of P2X4R in the microglia. The results of present study showed that mast cell activation markedly promoted the expression of P2X4R and BDNF in microglial cells, which significantly enhanced the release of BDNF from microglial cells upon exposure to adenosine triphosphate. Mast cell-derived tryptase activated PAR2 that resulted in promoting the expression of P2X4R in microglial cells. Pretreatment with antibodies against tryptase or PAR2, or using tryptase-deficient HMC-1 cells or PAR2-deficient microglial cells abolished the increase in P2X4R expression and BDNF release. Increase in mitogen activated protein kinase phosphorylation was observed in the processes of mast cell-induced BDNF release and P2X4R expression. We conclude that mast cell activation has the capacity to promote the expression of P2X4R and BDNF in microglial cells.

Uridine 5'-triphosphate (UTP) protects against cerebral ischemia reperfusion injury in rats.

AIMS: To test the hypothesis that uridine 5'-triphosphate (UTP) had a protective effect on cerebral ischemia reperfusion (IR) injury in rats. METHODS: Ischemia was induced by intraluminal suture of middle cerebral artery occlusion (MCAO). UTP solution was delivered through an indwelling tail venous catheter via microinfusion pump 30 min after the occlusion of MCA at a rate of 0.5 ml/100 g/min. Neurological deficit score (NDS) and brain water content were determined 24 h after reperfusion. Infarct volume was determined by 2,3,5-triphenyl-tetrazolium chloride (TTC) staining and magnetic resonance imaging (MRI), and nerve cell death was studied under an electron microscope. RESULTS: There was a dose-dependent relationship among 10, 30 and 90 microg/kg UTP. The 90 microg/kg UTP had the best protective effect among the 3 groups. We compared 90 microg/kg UTP group with normal saline group and found that UTP had a protective effect on cerebral IR by the results of TTC staining (15.9% vs 30.5%, P<0.01). MRI at 6, 30 and 54 h after reperfusion showed smaller infarct volume in 90 microg/kg group compared with 0 microg/kg group (283.5, 352.1, 367.45 mm(3) vs 401.36, 576.75 and 677.11 mm(3), respectively), and electron microscope showed less nerve cell death in 90 microg/kg group compared with 0 microg/kg group. CONCLUSION: UTP has a dose-dependent protective effect on cerebral IR.