Indole-3-Propionic Acid, a Gut Microbiota Metabolite, Protects Against the Development of Postoperative Delirium.

OBJECTIVE: The aim was to determine preoperative gut microbiota metabolites that may be associated with postoperative delirium (POD) development in patients and further study in rodents. SUMMARY BACKGROUND DATA: POD occurs in 9% to 50% of older patients undergoing anesthesia/surgery but lacks effective treatments or prevention. High-throughput metabolomics using liquid chromatography with tandem mass spectrometry has accelerated disease-related biomarkers discovery. We performed metabolomic studies in humans to identify potential metabolite biomarkers linked to POD and examined potential mechanisms in rodents. METHODS: We performed a prospective observational cohort study to examine the metabolomic changes that were associated with the development of POD. Then the gut microbiota-related metabolomic changes were recapitulated by gut microbiota perturbation in rodents. POD was assessed in mice using a battery of behavioral tests including novel objective test, Y-maze test, open-field test, and buried food test. The mechanisms through which gut microbiota-related metabolomic changes influenced POD were examined using chemogenetics. RESULTS: Indole-3-propionic acid (IPA) is a gut microbiota metabolite that belongs to the indole family. Baseline plasma levels of IPA were significantly inversely correlated with the onset of POD in 103 (17 cases) human individuals. This relationship was validated in preclinical mouse models for POD: reducing IPA levels through gut microbiota perturbation promoted POD-like behavior. More importantly, IPA administration deterred POD-like behavior. Colonization of germ-free mice with mutant Clostridium sporogenes that did not produce IPA-promoted POD-like behavior. Chemogenetic studies revealed that the protective effect of IPA in mice was mediated, in part, by peroxisome proliferator-activated receptor gamma coactivator 1-alpha in hippocampal interneurons. CONCLUSIONS: Gut microbiota-derived IPA is an important molecule implicated in the pathogenesis of POD, which could potentially be harnessed for POD prevention.

The nonopioid cholinergic agonist GTS-21 mitigates morphine-induced aggravation of burn injury pain together with inhibition of spinal microglia activation in young rats.

BACKGROUND: Repetitive opioid use does not always alleviate basal pain, procedural pain, or both after burn injury. Mitigation of burn injury-site pain can be achieved by GTS-21 stimulation of α7-acetylcholine nicotinic receptors (α7AChRs) and reduced microglia activation in rat. We tested the hypothesis that morphine exaggerates burn injury-site pain and GTS-21 alleviates both morphine-induced aggravated burn injury pain and microglia activation. METHODS: Young rats with dorsal paw burn injury or sham-burn received intraperitoneal saline, morphine, GTS-21, or a combination twice daily for 14 days. Ipsilateral plantar pain thresholds were tested every other day before morning drugs from days 0-20. Spinal microglia activation, evidenced as pain-transducer (tumour necrosis factor-α [TNF-α], interleukin [IL]-6, IL-1ß, nuclear factor kappa B [NF-κB], Toll-like receptor 4 [TLR4]) expression, was examined using immunohistochemistry and immunoblot. In cultured microglia, morphine-induced cytokine expression was measured (quantitative polymerase chain reaction/enzyme-linked immunosorbent assay [qPCR/ELISA]). RESULTS: Morphine aggravated allodynia at day 5 in sham-burn (P=0.039, n=8-11) but significantly aggravated burn injury site allodynia by day 3 (P=0.010, n=8-11). Microgliosis paralleled nociceptive behaviour changes where burn injury with morphine had highest microgliosis compared with burn injury, morphine alone, or controls (number of cells per field [SD]: 33.8 [2.4], 18.0 [4.1], 8.2 [1.9], and 4.8 [2.0], respectively; P<0.001, n=4-5]. GTS-21 reversed the morphine-induced pain component in sham-burn and burn injury rats together with reduced microgliosis and spinal pain-transducer expression (TNF-α, IL-6, IL-1ß, NF-κB, and TLR4). Morphine-exposed microglial cells showed increased cytokine expression, which was mitigated by GTS-21. CONCLUSIONS: Morphine or burn injury alone increases pain together with microgliosis and pain-transducer expression. Morphine administration augments burn injury-site nociception sooner and aggravated spinal microgliosis and inflammatory pain-transducer expression. GTS-21 has the potential to treat morphine-induced pain in burn injury.

Nonopioid GTS-21 Mitigates Burn Injury Pain in Rats by Decreasing Spinal Cord Inflammatory Responses.

BACKGROUND: Burn injury (BI) pain consists of inflammatory and neuropathic components and activates microglia. Nicotinic alpha 7 acetylcholine receptors (α7AChRs) expressed in microglia exhibit immunomodulatory activity during agonist stimulation. Efficacy of selective α7AChR agonist GTS-21 to mitigate BI pain and spinal pain-mediators was tested. METHODS: Anesthetized rats after hind-paw BI received intraperitoneal GTS-21 or saline daily. Allodynia and hyperalgesia were tested on BI and contralateral paw for 21 days. Another group after BI receiving GTS-21 or saline had lumbar spinal cord segments harvested (day 7 or 14) to quantify spinal inflammatory-pain transducers or microglia activation using fluorescent marker, ionized calcium-binding adaptor protein (Iba1). RESULTS: BI significantly decreased allodynia withdrawal threshold from baseline of ~9-10 to ~0.5-1 g, and hyperalgesia latency from ~16-17 to ~5-6 seconds by day 1. Both doses of GTS-21 (4 or 8 mg/kg) mitigated burn-induced allodynia from ~0.5-1 to ~2-3 g threshold (P = .089 and P = .010), and hyperalgesia from ~5-6 to 8-9 seconds (P < .001 and P < .001) by day 1. The GTS-21 group recovered to baseline pain threshold by day 15-17 compared to saline-treated, where the exaggerated nociception persisted beyond 15-17 days. BI significantly (P < .01) increased spinal cord microgliosis (identified by fluorescent Iba1 staining), microglia activation (evidenced by the increased inflammatory cytokine), and pain-transducer (protein and/or messenger RNA [mRNA]) expression (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß], nuclear factor-kappa B [NF-κB], interleukin-6 [IL-6], Janus-associated kinase signal transducer and activator of transcription 3 [JAK-STAT3], and/or N-methyl-D-aspartate receptor [NMDAR]). GTS-21 mitigated pain-transducer changes. The α7AChR antagonist methyllycaconitine nullified the beneficial effects of GTS-21 on both increased nociception and pain-biomarker expression. CONCLUSIONS: Nonopioid, α7AChR agonist GTS-21 elicits antinociceptive effects at least in part by decreased activation spinal-cord pain-inducers. The α7AChR agonist GTS-21 holds promise as potential therapeutic adjunct to decrease BI pain by attenuating both microglia changes and expression of exaggerated pain transducers.

Gut Microbiota Influences Neuropathic Pain Through Modulating Proinflammatory and Anti-inflammatory T Cells.

BACKGROUND: Gut microbiota, a consortium of diverse microorganisms residing in the gastrointestinal tract, has emerged as a key player in neuroinflammatory responses, supporting the functional relevance of the "gut-brain axis." Chronic-constriction injury of the sciatic nerve (CCI) is a commonly used animal model of neuropathic pain with a major input from T cell-mediated immune responses. In this article, we sought to examine whether gut microbiota influences CCI neuropathic pain, and, if so, whether T-cell immune responses are implicated. METHODS: We used a mixture of wide-spectrum oral antibiotics to perturbate gut microbiota in mice and then performed CCI in these animals. Nociceptive behaviors, including mechanical allodynia and thermal hyperalgesia, were examined before and after CCI. Additionally, we characterized the spinal cord infiltrating T cells by examining interferon (IFN)-γ, interleukin (IL)-17, and Foxp3. Using a Foxp3-GFP-DTR "knock-in" mouse model that allows punctual depletion of regulatory T cells, we interrogated the role of these cells in mediating the effects of gut microbiota in the context of CCI neuropathic pain. RESULTS: We found that oral antibiotics induced gut microbiota changes and attenuated the development of CCI neuropathic pain, as demonstrated by dampened mechanical allodynia and thermal hyperalgesia. Percentages of IFN-γ-producing Th1 cells and Foxp3+ regulatory T cells were significantly different between animals that received oral antibiotics (Th1 mean = 1.0, 95% confidence interval [CI], 0.9-1.2; Foxp3 mean = 8.1, 95% CI, 6.8-9.3) and those that received regular water (Th1 mean = 8.4, 95% CI, 7.8-9.0, P < .01 oral antibiotics versus water, Cohen's d = 18.8; Foxp 3 mean = 2.8, 95% CI, 2.2-3.3, P < .01 oral antibiotics versus water, Cohen's d = 6.2). These T cells characterized a skewing from a proinflammatory to an anti-inflammatory immune profile induced by gut microbiota changes. Moreover, we depleted Foxp3+ regulatory T cells and found that their depletion reversed the protection of neuropathic pain mediated by gut microbiota changes, along with a dramatic increase of IFN-γ-producing Th1 cell infiltration in the spinal cord (before depletion mean = 2.8%, 95% CI, 2.2-3.5; after depletion mean = 9.1%, 95% CI, 7.2-11.0, p < .01 before versus after, Cohen's d = 5.0). CONCLUSIONS: Gut microbiota plays a critical role in CCI neuropathic pain. This role is mediated, in part, through modulating proinflammatory and anti-inflammatory T cells.

Proliferator-Activated Receptor-Gamma Coactivator-1α Haploinsufficiency Promotes Pain Chronification After Burn Injury.

BACKGROUND: Tissue injuries such as surgery and trauma are usually accompanied by simultaneous development of acute pain, which typically resolves along with tissue healing. However, in many cases, acute pain does not resolve despite proper tissue repair; rather, it transitions to chronic pain. In this study, we examined whether proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a master regulator of mitochondria biogenesis, is implicated in pain chronification after burn injury in mice. METHODS: We used PGC-1α and littermates PGC-1α mice of both sex. Burn injury was induced on these mice. Hindpaw mechanical withdrawal thresholds and thermal withdrawal latency were examined. RESULTS: Hindpaw mechanical withdrawal thresholds and thermal withdrawal latencies were comparable at baseline between PGC-1α and PGC-1α mice. After burn injury, both PGC-1α and PGC-1α mice exhibited an initial dramatic decrease of withdrawal parameters at days 3 and 5 after injury. While PGC-1α mice fully recovered their withdrawal parameters to preinjury levels by days 11-14, PGC-1α mice failed to recover those parameters during the same time frame, regardless of sex. Moreover, we found that PGC-1α mice resolved tissue inflammation in a similar fashion to PGC-1α mice using a chemiluminescence-based reactive oxygen species imaging technique. CONCLUSIONS: Taken together, our data suggest that PGC-1α haploinsufficiency promotes pain chronification after burn injury.

Methylphenidate and Morphine Combination Therapy in a Rat Model of Chronic Pain.

BACKGROUND: The incremental dose of opioids used in chronic pain management often leads to a reduced opioid analgesic effect, opioid misuse, and addiction. Central dopamine (DA) dysfunction contributes to the chronicity of pain and a decreased opioid analgesic effect. Methylphenidate (MPH/Ritalin) enhances central DA function by inhibiting DA reuptake. In this study, we used a rat model of chronic pain to examine whether combination of MPH with morphine (MOR) would improve the MOR analgesic effect under a chronic pain condition. METHODS: Tibiotarsal joint Complete Freund's Adjuvant (CFA) injection in rats was utilized to induce chronic nociception. The analgesic effect of low-dose MPH (0.25 mg/kg), low-dose MOR (2.5 mg/kg), and their combination was examined in CFA rats. Nociceptive behavior was assessed by von Frey test. Conditioned place preference (CPP) and open field tests (OFTs) were used to examine the rewarding behavior and locomotor activity in rats, respectively. RESULTS: Our findings are as follows: (1) in CFA rats with chronic pain, 2.5 mg/kg of MOR had less analgesic effect than 10 mg/kg of MOR at 28 days after injury (95% confidence intervals [CIs] for difference of means of von Frey threshold in gram: -11.9 [-6.5 to -17.3]); (2) in the 1-hour time window of 30-90 minutes after injection, the combination of MPH (0.25 mg/kg) with MOR (2.5 mg/kg) increased synergistically and prolonged the analgesic effect in CFA rats as compared with MPH or MOR alone (P = .01 for MPH by MOR interaction, and 95% CIs for difference of means of von Frey threshold in gram: 3.3 [1.37-6.12] for the combination versus MPH and 3.2 [1.35-5.74] for the combination versus MOR); (3) at the low dose (0.25 mg/kg), MPH did not increase locomotor activity (MOR + MPH versus MOR, P = .13) nor significantly enhanced MOR reward behavior (MOR + MPH versus MOR, P = .63) in CFA rats. CONCLUSIONS: Our data suggest that a combination therapy using low-dose MPH and MOR may produce a MOR-sparing effect in chronic pain management.

Persistent Nociception Facilitates the Extinction of Morphine-Induced Conditioned Place Preference.

BACKGROUND: As opioid abuse and addiction have developed into a major national health crisis, prescription of opioids for pain management has become more controversial. However, opioids do help some patients by providing pain relief and improving the quality of life. To better understand the addictive properties of opioids under chronic pain conditions, we used a conditioned place preference (CPP) paradigm to examine the rewarding properties of morphine in rats with persistent nociception. METHODS: Spared nerve injury (SNI) model was used to induce persistent nociception in rats. Nociceptive behavior was assessed by von Frey test. CPP test was used to examine the rewarding properties of morphine. RESULTS: Our findings are as follows: (1) SNI rats did not show a difference compared with sham rats in magnitude of morphine-induced CPP 1 day after last morphine injection (2-way analysis of variance; for SNI versus sham, F[1,42] = 0.014, P = .91; and 95% confidence intervals for difference of means, -5.9 [-58 to 46], 0.76 [-51 to 53], and 0.90 [-51 to 53] for 2.5, 5, and 10 mg/kg, respectively); (2) increasing morphine dosage (2.5, 5, and 10 mg/kg) did not further increase the magnitude of CPP in both sham and SNI rats (for dosage: F[2,42] = 0.94, P = .40); and (3) morphine-induced CPP persisted in sham rats but extinguished in SNI rats when tested at 8 days after last morphine injection (for sham versus SNI: Bonferroni correction, P < .006 for both 5 and 10 mg/kg doses; and 95% confidence intervals for difference of means, 80.3 [19.7-141] and 87.0 [26.3-148] for 5 and 10 mg/kg, respectively). CONCLUSIONS: Our data provide new evidence supporting the notion that the brain's reward circuitry changes in the context of persistent pain. This observational study suggests that future investigation into the neurobiology of opioid reward requires consideration of the circumstances in which opioid analgesics are administered.

Ultrasmall Superparamagnetic Iron Oxide Imaging Identifies Tissue and Nerve Inflammation in Pain Conditions.

Objective: Correlation between radiologic structural abnormalities and clinical symptoms in low back pain patients is poor. There is an unmet clinical need to image inflammation in pain conditions to aid diagnosis and guide treatment. Ferumoxytol, an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle, is clinically used to treat iron deficiency anemia and showed promise in imaging tissue inflammation in human. We explored whether ferumoxytol can be used to identify tissue and nerve inflammation in pain conditions in animals and humans. Methods: Complete Freud's adjuvant (CFA) or saline was injected into mice hind paws to establish an inflammatory pain model. Ferumoxytol (20 mg/kg) was injected intravenously. Magnetic resonance imaging (MRI) was performed prior to injection and 72 hours postinjection. The changes in the transverse relaxation time (T2) before and after ferumoxytol injection were compared between mice that received CFA vs saline injection. In the human study, we administered ferumoxytol (4 mg/kg) to a human subject with clinical symptoms of lumbar radiculopathy and compared the patient with a healthy subject. Results: Mice that received CFA exhibited tissue inflammation and pain behaviors. The changes in T2 before and after ferumoxytol injection were significantly higher in mice that received CFA vs saline (20.8 ± 3.6 vs 2.2 ± 2.5, P = 0.005). In the human study, ferumoxytol-enhanced MRI identified the nerve root corresponding to the patient's symptoms, but the nerve root was not impinged by structural abnormalities, suggesting the potential superiority of this approach over conventional structural imaging techniques. Conclusions: Ferumoxytol-enhanced MRI can identify tissue and nerve inflammation and may provide a promising diagnostic tool in assessing pain conditions in humans.

Nortriptyline Enhances Morphine-Conditioned Place Preference in Neuropathic Rats: Role of the Central Noradrenergic System.

BACKGROUND: Combination drug therapy is commonly used to treat chronic pain conditions such as neuropathic pain, and antidepressant is often used together with opioid analgesics. While rewarding is an intrinsic property of opioid analgesics, it is unknown whether the use of an antidepressant would influence opioid reward, which may contribute to opioid addiction. In this study, we examined whether nortriptyline (a tricyclic antidepressant and a first-line medication for neuropathic pain) would enhance the morphine rewarding property in both naive and chronic constriction sciatic nerve injury (CCI) rats. METHODS: The rewarding effect of these drugs was assessed using conditioned place preference (CPP). The real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay analysis were used to investigate the function of central noradrenergic system. RESULTS: In naive rats, coadministration of nortriptyline with morphine did not change the acquisition of morphine-induced CPP. However, nortriptyline enhanced the acquisition, delayed the extinction, and augmented the reinstatement of morphine-induced CPP in CCI rats. In CCI rats treated with both nortriptyline and morphine, the expression of α2A-adrenergic receptors, norepinephrine transporter, and tyrosine hydroxylase was markedly decreased in the locus coeruleus, whereas the norepinephrine concentration in the nucleus accumbens was remarkably increased. CONCLUSIONS: These results demonstrate that nortriptyline enhanced morphine reward when both drugs were used to treat neuropathic pain in rats and that this behavioral phenotype is likely to be mediated by upregulation of the central noradrenergic system. These findings may have implications in opioid therapy commonly used for chronic pain management.

MRI reveals differential effects of amphetamine exposure on neuroglia in vivo.

How amphetamine affects the neuroglia in living brains is not well understood. In an effort to elucidate this effect, we investigated neuroglia in response to amphetamine exposure using antisense (AS) or sense (S) phosphorothioate-modified oligodeoxynucleotide (sODN) sequences that correspond to glial fibrillary acidic protein (GFAP) mRNA (AS-gfap or S-gfap, respectively) expression. The control is a random-sequence sODN (Ran). Using cyanine 5.5-superparamagnetic iron oxide nanoparticle (Cy5.5-SPION) labeling and fluorescent microscopy, we demonstrated that living neural progenitor cells (PC-12.1), as well as the cells in fresh brain slices and intact brains of male C57BL6 mice, exhibited universal uptake of all of the sODNs but rapidly excluded all sODN-Ran and most S-gfap. Moreover, transmission electron microscopy revealed electron-dense nanoparticles only in the neuroglia of normal or transgenic mice [B6;DBA-Tg(Fos-tTA, Fos-EGFP*)1MmayTg(tetO-lacZ,tTA*)1Mmay/J] that had been administered AS-gfap or Cy5.5-SPION-gfap. Subtraction R2* maps from mice with acute and chronic amphetamine exposure demonstrated, validated by postmortem immunohistochemistry, a reduction in striatal neuroglia, with gliogenesis in the subventricular zone and the somatosensory cortex in vivo. The sensitivity of our unique gene transcript targeted MRI was illustrated by a positive linear correlation (r(2)=1.0) between in vivo MRI signal changes and GFAP mRNA copy numbers determined by ex vivo quantitative RT-PCR. The study provides direct evidence for targeting neuroglia by antisense DNA-based SPION-gfap that enables in vivo MRI of inaccessible tissue with PCR sensitivity. The results enable us to conclude that amphetamine induces toxicity to neuroglia in vivo, which may cause remodeling or reconnectivity of neuroglia.

Noninvasive detection of neural progenitor cells in living brains by MRI.

The presence of pericytes in brain regions undergoing repair is evident of the recruitment of bone marrow-derived multipotent regenerative cells to the neurovascular unit during angiogenesis. At present, post mortem sampling is the only way to identify them. Therefore, such cell typing is inadequate for preserving neural progenitor cells for any meaningful stem cell therapy. We aimed to target cerebral pericytes in vivo using dual gene transcript-targeted MRI (GT-tMRI) in male C57black6 mice after a 60-min bilateral carotid artery occlusion (BCAO). We attached superparamagnetic iron oxide nanoparticles (SPIONs) to phosphorothioate-modified micro-DNA that targets actin or nestin mRNA. Because BCAO compromises the blood-brain barrier (BBB) and induces expression of α-smooth muscle (αSM)-actin and nestin antigens by pericytes in new vessels, we delivered pericyte-specific magnetic resonance contrast agents (SPION-actin or SPION-nestin at 4 mg Fe/kg) by i.p. injection to C57black6 mice that had experienced BCAO. We demonstrated that the surge in cerebral iron content by inductively coupled plasma-mass spectrometry matched the increase in the frequency of relaxivity. We also found that SPION-nestin was colocalized in αSM- actin- and nestin-expressing pericytes in BCAO-treated C57black6 or transgenic mice [B6.Cg-Tg(CAG-mRFP1) 1F1Hadj/J, expressing red fluorescent protein by actin promoter]. We identified pericytes in the repair patch in living brains after BCAO with a voxel size of 0.03 mm(3). The presence of electron-dense nanoparticles in vascular pericytes in the region of BBB injury led us to draw the conclusion that GT-tMRI can noninvasively reveal neural progenitor cells during vascularization.

Highly synchronized cortical circuit dynamics mediate spontaneous pain in mice.

Cortical neural dynamics mediate information processing for the cerebral cortex, which is implicated in fundamental biological processes such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes the cortical network into an aberrant state and, if so, whether it can be brought back to a "normal" operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with spontaneous pain-like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos-expressing neurons and selective activation of GABAergic interneurons significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More important, restoring a "normal" range of neural dynamics through attenuation of pain-induced S1 synchronization alleviated pain-like behavior. These results suggest that spontaneous pain pushed the S1 regional network into a synchronized state, whereas reversal of this synchronization alleviated pain.

Role of 5-HT1A-mediated upregulation of brain indoleamine 2,3 dioxygenase 1 in the reduced antidepressant and antihyperalgesic effects of fluoxetine during maintenance treatment.

The reduced antidepressant and antihyperalgesic effects of selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine during maintenance treatment has been reported, but little is known about the molecular mechanism of this phenomenon. In three comorbid pain and depression animal models (genetic predisposition, chronic social stress, arthritis), we showed that the fluoxetine's antidepressant and antihyperalgesic effects were reduced during the maintenance treatment. Fluoxetine exposure induced upregulation of the 5-hydroxytryptamine 1A (5-HT1A) auto-receptor and indoleamine 2,3 dioxygenase 1 (IDO1, a rate-limiting enzyme of tryptophan metabolism) in the brainstem dorsal raphe nucleus (DRN), which shifted the tryptophan metabolism away from the 5-HT biosynthesis. Mechanistically, IDO1 upregulation was downstream to fluoxetine-induced 5-HT1A receptor expression because 1) antagonism of the 5-HT1A receptor with WAY100635 or 5-HT1A receptor knockout blocked the IDO1 upregulation, and 2) inhibition of IDO1 activity did not block the 5-HT1A receptor upregulation following fluoxetine exposure. Importantly, inhibition of either the 5-HT1A receptor or IDO1 activity sustained the fluoxetine's antidepressant and antihyperalgesic effects, indicating that 5-HT1A-mediated IDO1 upregulation in the brainstem DRN contributed to the reduced antidepressant and antihyperalgesic effects of fluoxetine. These results suggest a new strategy to improving the therapeutic efficacy of SSRI during maintenance treatment.

Electroacupuncture attenuates surgical pain-induced delirium-like behavior in mice via remodeling gut microbiota and dendritic spine.

Surgical pain is associated with delirium in patients, and acupuncture can treat pain. However, whether electroacupuncture can attenuate the surgical pain-associated delirium via the gut-brain axis remains unknown. Leveraging a mouse model of foot incision-induced surgical pain and delirium-like behavior, we found that electroacupuncture stimulation at specific acupoints (e.g., DU20+KI1) attenuated both surgical pain and delirium-like behavior in mice. Mechanistically, mice with incision-induced surgical pain and delirium-like behavior showed gut microbiota imbalance, microglia activation in the spinal cord, somatosensory cortex, and hippocampus, as well as an enhanced dendritic spine elimination in cortex revealed by two-photon imaging. The electroacupuncture regimen that alleviated surgical pain and delirium-like behavior in mice also effectively restored the gut microbiota balance, prevented the microglia activation, and reversed the dendritic spine elimination. These data demonstrated a potentially important gut-brain interactive mechanism underlying the surgical pain-induced delirium in mice. Pending further studies, these findings revealed a possible therapeutic approach in preventing and/or treating postoperative delirium by using perioperative electroacupuncture stimulation in patients.

Comparison between acupuncture therapy and gabapentin for chronic pain: a pilot study.

BACKGROUND: We examined whether the effect of true electroacupuncture on pain and functionality in chronic pain participants can be differentiated from that of medication (gabapentin) by analyzing quantitative sensory testing (QST). METHODS: We recruited chronic back and neck pain participants who received six sessions (twice weekly) of true electroacupuncture versus sham electroacupuncture or 3 weeks of gabapentin versus placebo treatment. QST profiles, pain scores, and functionality profile were obtained at baseline (visit 1) and after three sessions (visit 4) or six sessions (visit 7) of acupuncture or 3 weeks of gabapentin or placebo. RESULTS: A total of 50 participants were analyzed. We found no differences in QST profile changes (p = 0.892), pain reduction (p = 0.222), or functionality (p = 0.254) between the four groups. A major limitation of this pilot study was the limited number of study participants in each group. CONCLUSION: This pilot study suggests that a large-scale clinical study with an adequate sample size would be warranted to compare acupuncture and medication therapy for chronic pain management. TRIAL REGISTRATION NUMBER: NCT01678586 (ClinicalTrials.gov).

Diffusion-weighted magnetic resonance imaging reversal by gene knockdown of matrix metalloproteinase-9 activities in live animal brains.

The involvement of matrix metalloproteinase-9 (MMP-9) activities in the development of abnormal water diffusion in the brain after cardiac arrest is not fully understood. We used magnetic resonance imaging to determine the correlation between MMP-9 activity and the mechanism of abnormal water diffusion after global cerebral ischemia (GCI)-induced brain damage in C57black6 mice. We induced GCI in mice by occluding both carotid arteries for 60 min, then allowing reperfusion. We labeled a short DNA that targets mmp-9 mRNA activity [phosphorothioate-modified oligodeoxynucleotide (sODN)-mmp9] or a control probe without intracellular target (sODN-Ran) with iron-based MR contrast agent [superparamagnetic iron oxide nanoparticle (SPION)-mmp9 or SPION-Ran] or fluorescein isothiocyanate (FITC)-sODN-mmp9 or FITC-sODN-Ran; we then delivered these probes by intracerebroventricular infusion or intraperitoneal injection within 3 h of reperfusion. At low dose (120 pmol/kg) the SPION-mmp9 probe was retained at significant levels in the striatum and cortex of living brains 10 h after GCI. Probe retention was validated by similar elevation of mmp-9 mRNA and antigens in postmortem samples taken from regions that exhibited GCI-induced hyperintensity in diffusion-weighted imaging, and a significant reduction in apparent diffusion coefficient (rADC, p = 0.0006, n = 12). At a higher dose (120 nmol/kg), the FITC-sODN-mmp9 probe revealed significant knockdown of MMP-9 activity, per zymography, and a reversal of striatal rADC (p = 0.004, n = 6). These observations were not duplicated in the control group. We conclude that expression of mmp-9 mRNA is associated with abnormal ADC after GCI.

Noninvasive delivery of gene targeting probes to live brains for transcription MRI.

We aimed to test the feasibility of detecting gliosis in living brains when the blood-brain barrier (BBB) is disrupted. We designed a novel magnetic resonance (MR) probe that contains superparamagnetic iron oxide nanoparticles (SPION, a T2 susceptibility contrast agent) linked to a short DNA sequence complementary to the cerebral mRNA of glial fibrillary acidic protein (GFAP) found in glia and astrocytes. As a control, we also used a sequence complementary to the mRNA of beta-actin. Our objectives are to demonstrate that this new probe, SPION-gfap, could be delivered to the brain when administered by eyedrop solution to the conjunctival sac. We induced BBB leakage by puncture wound, global cerebral ischemia, and cortical spreading depression in C57BL6 mice; 1 day after probe delivery we acquired T2* MR images and R2* (R2* = 1/T2*) maps using a transcription MRI technique in live mice. We found that the SPION-gfap probe reported foci with elevated signal in subtraction R2* maps and that these foci matched areas identified as having extensive glial network (gliosis) in postmortem immunohistochemistry. Similarly, animals administered the control probe exhibited foci of R2* elevation that matched beta-actin-expressing endothelia in the vascular wall. We conclude that our modular MR probe, delivered in an eyedrop solution, effectively reports gliosis associated with acute neurological disorders in living animals. As BBB leakage is often observed in acute neurological disorders, this study also served to validate noninvasive delivery of MR probes to the brains of live animals after acute neurological disorders.

Alleviation of trigeminal neuropathic pain by electroacupuncture: the role of hyperpolarization-activated cyclic nucleotide-gated channel protein expression in the Gasserian ganglion.

INTRODUCTION: The aim of this study was to examine the effect of electroacupuncture (EA) on trigeminal neuropathic pain in rats and explore the potential mechanism underlying the putative therapeutic effect of EA. METHODS: Trigeminal neuropathic pain behavior was induced in rats by unilateral chronic constriction injury of the distal infraorbital nerve (dIoN-CCI). EA was administered at ST2 (Sibai) and Jiachengjiang. A total of 60 Sprague Dawley rats were divided into the following four groups (n = 15 per group) to examine the behavioral outcomes after surgery and/or EA treatment: sham (no ligation); dIoN-CCI (received isoflurane only, without EA treatment); dIoN-CCI+EA-7d (received EA treatment for 7 days); and dIoN-CCI+EA-14d (received EA treatment for 14 days). Both evoked and spontaneous nociceptive behaviors were measured. Of these, 12 rats (n = 4 from sham, dIoN-CCI, and dIoN-CCI+EA-14d groups, respectively) were used to analyze protein expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel in the Gasserian ganglion (GG) by immunohistochemistry. RESULTS: dIoN-CCI rats exhibited mechanical allodynia and increased face-grooming activity that lasted at least 35 days. EA treatment reduced mechanical allodynia and face-grooming in dIoN-CCI rats. Overall, 14 days of EA treatment produced a prolonged anti-nociceptive effect as compared to 7-day EA treatment. The counts of HCN1 and HCN2 immunopositive puncta were increased in the ipsilateral GG in dIoN-CCI rats and were reduced by 14 days of EA treatment. DISCUSSION: EA treatment relieved trigeminal neuropathic pain in dIoN-CCI rats, and this effect was dependent on the duration of EA treatment. The downregulation of HCN expression may contribute to the anti-nociceptive effect of EA in this rat model of trigeminal neuropathic pain.

A randomized trial to assess the immediate impact of acupuncture on quantitative sensory testing, pain, and functional status.

In this randomized clinical trial, we examined whether the effect of true acupuncture can be differentiated from sham acupuncture (pain and functionality) by analyzing quantitative sensory testing (QST) profiles in chronic pain participants. We recruited 254 healthy or chronic back and neck pain participants. Healthy subjects were included to control for a possible effect of acupuncture on baseline QST changes. Study participants received 6 sessions (twice weekly) of true acupuncture, sham acupuncture, or no acupuncture treatment (routine care). Quantitative sensory testing profiles, pain scores, and functionality profile were obtained at baseline (visit 1) and after 3 (visit 4) or 6 sessions (visit 7). A total of 204 participants were analyzed. We found no QST profile changes among 3 groups (P = 0.533 and P = 0.549, likelihood ratio tests) in either healthy or chronic pain participants. In chronic back and neck pain participants, true acupuncture reduced pain (visit 4: difference in mean [DIM] = -0.8, 95% confidence interval [CI]: -1.4 to -0.1, adjusted P = 0.168; visit 7: DIM = -1.0, 95% CI: -1.7 to -0.3, adjusted P = 0.021) and improved functional status including physical functioning (DIM = 14.21, 95% CI: 5.84-22.58, adjusted P = 0.003) and energy/fatigue (DIM = 12.28, 95% CI: 3.46-21.11, adjusted P = 0.021) as compared to routine care. Our results indicate that QST was not helpful to differentiate between true acupuncture and sham acupuncture (primary outcome) in this study, although true acupuncture reduced pain and improved functionality (secondary outcomes) when compared with routine care.

Mannose binding lectin gene deficiency increases susceptibility to traumatic brain injury in mice.

Mannose binding lectin (MBL) initiates complement activation and exacerbates tissue damage after systemic ischemia/reperfusion. We tested the hypothesis that MBL activates complement and worsens outcome using two levels of controlled cortical impact (CCI) in mice. After moderate CCI (0.6 mm depth), MBL immunostaining was detected on injured endothelial cells of wild-type (WT) mice and C3d was detected in MBL KO (deficient in MBL A/C) and WT mice, suggesting that MBL is dispensable for terminal complement activation after CCI. Brain neutrophils, edema, blood-brain barrier permeability, gross histopathology, and motor dysfunction were similar in injured MBL KO and WT mice. In mice subjected to mild CCI (0.2 mm), MBL KO mice had almost two-fold increased acute CA3 cell degeneration at 6 h (P<0.01 versus WT). Naive MBL KO mice had decreased brain volume but performed similar to WT mice in two distinct Morris water maze (MWM) paradigms. However, injured MBL KO mice had impaired performance in cued platform trials (P<0.05 versus WT), suggesting a transient nonspatial learning deficit in injured MBL KO mice. The data suggest that MBL deficiency increases susceptibility to CCI through C3-independent mechanisms and that MBL-deficient patients may be at increased risk of poor outcome after traumatic brain injury.