Lixian Xu

Post-injury administration of minocycline: An effective treatment for nerve-injury induced neuropathic pain

Neuropathic pain is an intractable clinical problem, affecting millions of people worldwide. Preemptive administration of minocycline has been confirmed useful for treating neuropathic pain by inhibiting spinal microglia activation and consequently lowering proinflammatory cytokine expression. However, most patients with neuropathic pain have no chance to receive preemptive treatment and it remains unclear whether there is a therapeutic time window for post treatment with minocycline. The present study is to confirm the effect and the therapeutic time window of intrathecal minocycline on spinal nerve ligation (SNL)-induced neuropathic pain after lesion. Behavioral test and immunohistochemistry are utilized to determine the variation of mechanical allodynia and microglia phosphorylated-p38 (p-p38) expression respectively after intrathecal minocycline. Results showed that post-injury intrathecal minocycline attenuated mechanical allodynia effectively together with inhibiting spinal microglia p-p38 expression on post operative day (POD) 1, POD 3 and POD 7. Additionally, results from POD 10 and POD 21 showed that intrathecal minocycline suppressed spinal microglia p-p38 expression but without any effects on reversing mechanical allodynia. It is concluded that post-injury intrathecal minocycline is an effective therapeutic intervention for treating SNL-induced neuropathic pain by inhibiting spinal microglia activation. Accordingly, there is indeed a therapeutic time window for post-injury intrathecal minocycline, which is the initiation stage of neuropathic pain development.

Neuronal NR2B-containing NMDA receptor mediates spinal astrocytic c-Jun N-terminal kinase activation in a rat model of neuropathic pain.

Spinal N-methyl d-aspartate receptor (NMDAR) plays a pivotal role in nerve injury-induced central sensitization. Recent studies suggest that NMDAR also contributes to neuron-astrocyte signaling. c-Jun N-terminal kinase (JNK) is persistently and specifically activated (indicated by phosphorylation) in spinal cord astrocytes after nerve injury and thus it is considered as a dependable indicator of pain-related astrocytic activation. NMDAR-mediated JNK activation in spinal dorsal horn might be an important form of neuron-astrocyte signaling in neuropathic pain. In the present study, we observed that intrathecal injection of MK-801, a noncompetitive NMDA receptor antagonist, or Ro25-6981 and ifenprodil, which are selective antagonists of NR2B-containing NMDAR each significantly reduced nerve injury-induced JNK activation. Double immunostaining showed that NR2B was highly expressed in neurons, indicating the effect of NMDAR antagonists on JNK activation was indirect. We further observed that intrathecal injection of NMDA (twice a day for 3 days) significantly increased spinal JNK phosphorylation. Besides, NMDAR-related JNK activation could be blocked by a neuronal nitric oxide synthase (nNOS) selective inhibitor (7-nitroindazole sodium salt) but not by a nNOS sensitive guanylyl cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). Finally, real-time RT-PCR and immunostaining showed that nerve injury-induced interleukin-1beta expression was dependent on astrocytic JNK activation. Treatments targeting NMDAR-nNOS pathway also influenced interleukin-1beta expression, which further confirmed our hypothesis. Taken together, our results suggest that neuronal NMDAR-nNOS pathway could activate astrocytic JNK pathway. Excitatory neuronal transmission initiates astrocytic activation-induced neuroinflammation in this way, which contributes to nerve injury-induced neuropathic pain.

Cationic amylose-encapsulated bovine hemoglobin as a nanosized oxygen carrier

Nanosized hemoglobin-based oxygen carriers are one of the most promising blood substitutes. In the present study, a comprehensive strategy for the preparation of nanosized cationic amylose-encapsulated hemoglobins (NCAHbs) was developed. First, cationic amylase (CA) was synthesized from amylose and quaternary ammonium salt by an etherification reaction. The structure of CA was characterized using Fourier transform infrared spectrophotometry (FTIR) and proton nuclear magnetic resonance spectrophotometry ((1)H NMR). The degree of substitution and the zeta potential were also measured. Then, the NCAHbs were prepared by electrostatic adhesion, reverse micelles and cross-linking. The UV-visible spectrophotometer was used to measure the entrapment efficiency (EE%) and drug loading efficiency (DL%) of the NCAHbs. Transmission electron microscopy and Malvern Nano-zs 90 analyzer were used to observe the size distribution and morphology of particles. Chemical structure was determined from the FTIR spectrum. A Hemox analyzer was used to measure the P(50) and Hill coefficients. A lethal hemorrhagic shock model in rats was used to evaluate the therapeutic effect of the NCAHbs. The results showed that the combined methods improved the size, stability, EE%, DL%, and oxygen-carrying capacity of the NCAHbs. The average diameter of the NCAHbs was 92.53xa0±xa03.64xa0nm, with a narrow polydispersity index of 0.027. The EE% was 80.05%xa0±xa01.56% and DL% was 61.55%xa0±xa01.41%. The P(50) and Hill coefficient were equal to 28.96xa0±xa01.33xa0mmHg and 2.55xa0±xa00.22, respectively. The size of NCAHbs remained below 200xa0nm for six days in PBS solution. The NCAHbs could effectively prevent lung injury from progressing to lethal hemorrhagic shock because they acted as both a volume expander and an oxygen carrier.

Effect of propofol on the levels of neurotransmitters in normal human brain: A magnetic resonance spectroscopy study

Though widely used in anesthesia for many years, the mechanism underlying propofol anesthesia on human is not clear. Animal studies have already demonstrated that propofol functioned mainly by affecting neurotransmitters release. In our study, 10 healthy volunteers ranging from 20 to 40 years old were enrolled. With the help of target-controlled infusion pump, propofol was delivered intravenously. The target-controlled concentration (TCC) of propofol was gradually elevated from 0.5 to 3.0 microg/ml (for 6 steps with an increment of 0.5 microg/ml). During each step the Observers Assessment of Alertness/Sedation Scale (OAA/S) was performed to evaluate asleep to awake/alert status. Magnetic resonance spectroscopy (MRS) was performed to evaluate neurotransmitters (choline compounds (Cho), creatine (Cr), glutamate (GLU), gamma-aminobutyric acid (GABA) and N-acetyl aspartate (NAA)) changes in brains following propofol anesthesia. OAA/S scoring showed that when the TCCs of propofol were 0, 1.5 and 3.0 microg/ml, the volunteers were in awake, sedative and unconscious, respectively. Significantly down-regulated Cho and GLU, but up-regulated GABA was observed in unconscious state in all the detected regions. NAA was decreased in unconscious status only in the hippocampus and thalamus. There was no obvious change in Cr levels in any statuses or brain regions. Our results indicate that propofol has an impact on the levels of neurotransmitters such as NAA, GLU, GABA and Cho in normal human brain. During propofol anesthesia, enhancement of inhibition or suppression of excitation may each play key roles in different brain regions.

Ketamine Depresses Toll-Like Receptor 3 Signaling in Spinal Microglia in a Rat Model of Neuropathic Pain

Reports suggest that microglia play a key role in spinal nerve ligation (SNL)-induced neuropathic pain, and toll-like receptor 3 (TLR3) has a substantial role in the activation of spinal microglia and the development of tactile allodynia after nerve injury. In addition, ketamine application could suppress microglial activation in vitro, and ketamine could inhibit proinflammatory gene expression possibly by suppressing TLR-mediated signal transduction. Therefore, the present study was designed to disclose whether intrathecal ketamine could suppress SNL-induced spinal microglial activation and exert some antiallodynic effects on neuropathic pain by suppressing TLR3 activation. Behavioral results showed that intrathecal ketamine attenuated SNL-induced mechanical allodynia, as well as spinal microglial activation, in a dose-dependent manner. Furthermore, Western blot analysis displayed that ketamine application downregulated SNL-induced phosphorylated-p38 (p-p38) expression, which was specifically expressed in spinal microglia but not in astrocytes or neurons. Besides, ketamine could reverse TLR3 agonist (polyinosine-polycytidylic acid)-induced mechanical allodynia and spinal microglia activation. It was concluded that intrathecal ketamine depresses TLR3-induced spinal microglial p-p38 mitogen-activated protein kinase pathway activation after SNL, probably contributing to the antiallodynic effect of ketamine on SNL-induced neuropathic pain.

Inhibition of spinal astrocytic c-Jun N-terminal kinase (JNK) activation correlates with the analgesic effects of ketamine in neuropathic pain

BackgroundWe have previously reported that inhibition of astrocytic activation contributes to the analgesic effects of intrathecal ketamine on spinal nerve ligation (SNL)-induced neuropathic pain. However, the underlying mechanisms are still unclear. c-Jun N-terminal kinase (JNK), a member of mitogen-activated protein kinase (MAPK) family, has been reported to be critical for spinal astrocytic activation and neuropathic pain development after SNL. Ketamine can decrease lipopolysaccharide (LPS)-induced phosphorylated JNK (pJNK) expression and could thus exert its anti-inflammatory effect. We hypothesized that inhibition of astrocytic JNK activation might be involved in the suppressive effect of ketamine on SNL-induced spinal astrocytic activation.MethodsImmunofluorescence histochemical staining was used to detect SNL-induced spinal pJNK expression and localization. The effects of ketamine on SNL-induced mechanical allodynia were confirmed by behavioral testing. Immunofluorescence histochemistry and Western blot were used to quantify the SNL-induced spinal pJNK expression after ketamine administration.ResultsThe present study showed that SNL induced ipsilateral pJNK up-regulation in astrocytes but not microglia or neurons within the spinal dorsal horn. Intrathecal ketamine relieved SNL-induced mechanical allodynia without interfering with motor performance. Additionally, intrathecal administration of ketamine attenuated SNL-induced spinal astrocytic JNK activation in a dose-dependent manner, but not JNK protein expression.ConclusionsThe present results suggest that inhibition of JNK activation may be involved in the suppressive effects of ketamine on SNL-induced spinal astrocyte activation. Therefore, inhibition of spinal JNK activation may be involved in the analgesic effects of ketamine on SNL-induced neuropathic pain.

Inhibiting astrocytic activation: a novel analgesic mechanism of ketamine at the spinal level?

Although ketamine is widely used as an analgesic agent and has an anti‐allodynic effect on neuropathic pain, the underlying analgesic mechanisms are not fully explained by the modern ‘neuronal‐based’ theories. As emerging studies have focused on the critical role of spinal astrocytes in the pathological pain states, we have hypothesized that there exist some ‘astrocytes‐related’ mechanisms in the analgesic function of ketamine. In the present study, using the spinal nerve ligation (SNL) pain model, we investigated the anti‐nociceptive effects of intraperitoneal or intrathecal ketamine on SNL‐induced neuropathic pain response, meanwhile, we investigated the astrocytic activation after ketamine administration on SNL rats. Behavioral data showed that either intraperitoneal or intrathecal ketamine inhibited SNL‐induced allodynia, however, immunohistochemistry showed that SNL induced astrocytic activation was suppressed by intrathecal but not intraperitoneal ketamine. Using quantitative Western blot analysis, our report showed that intrathecal ketamine down‐regulated glial fibrillary acidic protein expression, suggesting inhibition of SNL‐induced astrocytic activation, which wasn’t influenced by intraperitoneal administration. We conclude that intraperitoneal ketamine could alleviate SNL‐induced neuropathic pain via the classical ‘neuronal‐based’ mechanisms, but in addition, ‘astrocytes‐related’ mechanisms were also important underlying the anti‐allodynic effect of intrathecal ketamine.

The Inhibition of Spinal Astrocytic JAK2-STAT3 Pathway Activation Correlates with the Analgesic Effects of Triptolide in the Rat Neuropathic Pain Model

Neuropathic pain (NP) is an intractable clinical problem without satisfactory treatments. However, certain natural products have been revealed as effective therapeutic agents for the management of pain states. In this study, we used the spinal nerve ligation (SNL) pain model to investigate the antinociceptive effect of triptolide (T10), a major active component of the traditional Chinese herb Tripterygium wilfordii Hook F. Intrathecal T10 inhibited the mechanical nociceptive response induced by SNL without interfering with motor performance. Additionally, the anti-nociceptive effect of T10 was associated with the inhibition of the activation of spinal astrocytes. Furthermore, intrathecal administration of T10 attenuated SNL-induced janus kinase (JAK) signal transducers and activators of transcription 3 (STAT3) signalling pathway activation and inhibited the upregulation of proinflammatory cytokines, such as interleukin-6, interleukin-1 beta, and tumour necrosis factor-α, in dorsal horn astrocytes. Moreover, NR2B-containing spinal N-methyl D-aspartate receptor (NMDAR) was subsequently inhibited. Above all, T10 can alleviate SNL-induced NP via inhibiting the neuroinflammation in the spinal dorsal horn. The anti-inflammation effect of T10 may be related with the suppression of spinal astrocytic JAK-STAT3 activation. Our results suggest that T10 may be a promising drug for the treatment of NP.

Targeted delivery of neurogenin-2 protein in the treatment for cerebral ischemia-reperfusion injury.

Neurogenin-2 (Ngn2), as a proneural gene that promotes the survival and differentiation of neural precursor cells, is an attractive candidate for therapy against cerebral ischemia-reperfusion injury. However, the delivery approach limits its clinical application. To deliver Ngn2 protein into the cerebral ischemic region and exert a therapeutic effect on injured neurons after ischemia, we here reported that the fusion protein TAT-LBD-Ngn2 was constructed by fusing a transactivator of transcription (TAT) domain and a laminin-binding domain (LBD) to Ngn2. TAT-LBD-Ngn2 promoted the outgrowth of neuronal neurite, increased the survival rate and alleviated apoptosis of hippocampal neurons exposed to oxygen glucose deprivation inxa0vitro. Furthermore, a focal cerebral ischemia model in C57BL/6 mice showed that TAT-LBD-Ngn2 efficiently crossed the blood brain barrier, aggregated in the ischemic zone and was consistently incorporated into neurons. Moreover, TAT-LBD-Ngn2 transduced into brains attenuated neuronal degeneration and apoptosis in the ischemic zone. TAT-LBD-Ngn2 treatment resulted in a reduction of infarct volume that was associated with a parallel improvement in neurological functional outcomes after reperfusion. In conclusion, the targeted delivery of TAT-LBD-Ngn2 into the ischemic zone attenuated cerebral ischemia-reperfusion injury through the inhibition of neuronal degeneration and apoptosis, suggesting that TAT-LBD-Ngn2 is a promising target candidate for the treatment of ischemic stroke.

Combining ketamine with astrocytic inhibitor as a potential analgesic strategy for neuropathic pain. ketamine, astrocytic inhibitor and pain

BackgroundNeuropathic pain is an intractable clinical problem. Intrathecal ketamine, a noncompetitive N--methyl-D-aspartate receptor (NMDAR) antagonist, is reported to be useful for treating neuropathic pain in clinic by inhibiting the activity of spinal neurons. Nevertheless, emerging studies have disclosed that spinal astrocytes played a critical role in the initiation and maintenance of neuropathic pain. However, the present clinical therapeutics is still just concerning about neuronal participation. Therefore, the present study is to validate the coadministration effects of a neuronal noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine and astrocytic cytotoxin L-α-aminoadipate (LAA) on spinal nerve ligation (SNL)-induced neuropathic pain.ResultsIntrathecal ketamine (10, 100, 1000 μg/kg) or LAA (10, 50, 100 nmol) alleviated SNL-induced mechanical allodynia in a dose-dependent manner respectively. Phosphorylated NR1 (pNR1) or glial fibrillary acidic protein (GFAP) expression was down-regulated by intrathecal ketamine (100, 1000 μg/kg) or LAA (50, 100 nmol) respectively. The combination of ketamine (100 μg/kg) with LAA (50 nmol) showed superadditive effects on neuropathic pain compared with that of intrathecal administration of either ketamine or LAA alone. Combined administration obviously relieved mechanical allodynia in a quick and stable manner. Moreover, down-regulation of pNR1 and GFAP expression were also enhanced by drugs coadministration.ConclusionsThese results suggest that combining NMDAR antagonist ketamine with an astrocytic inhibitor or cytotoxin, which is suitable for clinical use once synthesized, might be a potential strategy for clinical management of neuropathic pain.