Meta-epidemiologic review: blinding and sham treatment in clinical trial design for osteopathic manipulative treatment research.

Objective: To analyze the consistency of study designs in osteopathic manipulative treatment (OMT) research, focusing on blinding protocols and the use of sham treatments. Data Source and Study Selection: PubMed and CINAHL were searched in January 2022. A total of 83 research studies between 2009 and 2021 were selected based on the presence of a double- or single-blind study design and/or sham treatment. Data Extraction and Analysis: Data regarding the primary outcome measures, blinding design, measures used to determine success of blinding, osteopathic technique used, and sham technique used for each eligible study were extracted and compared among different study designs. Results: A total of 5968 subjects participated in the 83 trials. The study population mainly consisted of asymptomatic individuals (25%) and chronic back pain patients (19%). Light touch was employed most commonly (49%) as the sham treatment, followed by unrelated sham (20%) and incomplete maneuvers (20%). Most studies blinded the subjects (80%) or the outcome evaluator/data analyzer (71%), while only 20% studies blinded the osteopathic physicians. Conclusions: Strict double-blinding is achievable for OMT clinical research by blinding the subjects and data collectors/analyzers rather than the osteopaths providing the actual treatment. The use of questionnaires to determine the success of blinding should be considered. Additionally, including OMT-naïve subjects is preferred to enhance blinding success. When designing a sham treatment, careful consideration should be given to blinding the data collector, accounting for the placebo effect, and incorporating an additional no-treatment control group to improve the rigor of the study design.

Microglial inflammation modulates opioid analgesic tolerance.

As we all know, opioids are the drugs of choice for treating severe pain. However, very often, opioid use leads to tolerance, dependence, and hyperalgesia. Therefore, understanding the mechanisms underlying opioid tolerance and designing strategies for increasing the efficacy of opioids in chronic pain are important areas of research. Microglia are brain macrophages that remove debris and dead cells from the brain and participate in immune defense of the central nervous system during an insult or injury. However, recent studies indicate that microglial activation and generation of proinflammatory molecules (e.g., cytokines, nitric oxide, eicosanoids, etc.) in the brain may contribute to opioid tolerance and other side effects of opioid use. In this review, we will summarize the evidence and possible mechanisms by which proinflammatory molecules produced by activated microglia may antagonize the analgesic effect induced by opioids, and thus, lead to opioid tolerance. We will also delineate specific examples of studies that suggest therapeutic targets to counteract the development of tolerance clinically using suppressors of microglial inflammation.

Chronicling changes in the somatosensory neurons after peripheral nerve injury.

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.

Inhibition of experimental visceral pain in rodents by cebranopadol.

The aim of this study was to investigate the efficacy of cebranopadol in two rodent models of visceral pain. Cebranopadol is a first-in-class analgesic with agonist activity at the nociceptin/orphanin FQ opioid peptide receptor and classical µ-, δ- and κ-opioid peptide receptors. Colitis was induced in Naval Medical Research Institute mice by intra-rectal infusion of mustard oil. The effects of intravenous cebranopadol pretreatment on spontaneous pain behaviours and referred allodynia and hyperalgesia were assessed. Pancreatitis was induced in Sprague-Dawley rats by intravenous administration of dibutyltin dichloride. After 6 days, the effects of intravenous cebranopadol on withdrawal reactions to mechanical abdominal stimulation with von Frey filaments were assessed. In mice with experimental colitis, cebranopadol dose-dependently inhibited spontaneous pain behaviours and allodynic and hyperalgesic withdrawal reactions, with half-maximal effective dose values of 4.6 µg/kg [95% confidence interval (CI): 2.9-7.9] for inhibition of spontaneous pain behaviours, 2.2 µg/kg (95% CI: 1.3-3.4) for inhibition of referred allodynia and 2.4 µg/kg (95% CI: 1.4-3.6) for inhibition of referred hyperalgesia in mice with colitis. In rats with experimental pancreatitis, cebranopadol dose-dependently inhibited abdominal tactile allodynia (half-maximal effective dose, 0.13 µg/kg; 95% CI: 0.03-0.49). Behavioural manifestations of visceral pain were almost completely abolished at the highest doses tested in mice (17.2 µg/kg, intravenous) and rats (2.4 µg/kg, intravenous). We conclude that cebranopadol is a potent and effective antiallodynic and antihyperalgesic agent in rodent models of visceral pain.

Anatomy and immunochemical characterization of the non-arterial peptidergic diffuse dural innervation of the rat and Rhesus monkey: Implications for functional regulation and treatment in migraine.

Objective The interplay between neuronal innervation and other cell types underlies the physiological functions of the dura mater and contributes to pathophysiological conditions such as migraine. We characterized the extensive, but understudied, non-arterial diffuse dural innervation (DDI) of the rat and Rhesus monkey. Methods We used a comprehensive integrated multi-molecular immunofluorescence labeling strategy to extensively profile the rat DDI and to a lesser extent that of the Rhesus monkey. Results The DDI was distributed across a dense, pervasive capillary network and included free nerve endings of peptidergic CGRP-expressing C fibers that were closely intertwined with noradrenergic (NA) sympathetic fibers and thin-caliber nonpeptidergic "C/Aδ" fibers. These newly identified C/Aδ fibers were unmyelinated, like C fibers, but expressed NF200, usually indicative of Aδ fibers, and uniquely co-labeled for the CGRP co-receptor, RAMP1. Slightly-larger caliber NF200-positive fibers co-labeled for myelin basic protein (MBP) and terminated as unbranched corpuscular endings. The DDI peptidergic fibers co-labeled for the lectin IB4 and expressed presumably excitatory α1-adrenergic receptors, as well as inhibitory 5HT1D receptors and the delta opioid receptor (δOR), but rarely the mu opioid receptor (µOR). Labeling for P2X3, TRPV1, TRPA1, and parasympathetic markers was not observed in the DDI. Interpretation These results suggest potential functional interactions, wherein peptidergic DDI fibers may be activated by stress-related sympathetic activity, resulting in CGRP release that could be detected in the circulation. CGRP may also activate nonpeptidergic C/Aδ fibers that are likely mechanosensitive or polymodal, leading to activation of post-synaptic pain transmission circuits. The distribution of α1-adrenergic receptors, RAMP1, and the unique expression of the δOR on CGRP-expressing DDI fibers suggest strategies for functional modulation and application to therapy.

Prevention of stress- or nitric oxide donor-induced medication overuse headache by a calcitonin gene-related peptide antibody in rodents.

Objective The objective of this study was the determination of the role of calcitonin gene-related peptide (CGRP) in the induction of medication overuse headache (MOH)-related migraine in an injury-free preclinical model. Methods Rats were primed by a 7-day period of exposure to acute migraine therapies including sumatriptan and morphine. After an additional 14-day drug-free period, rats were exposed to putative migraine triggers including bright light stress (BLS) or nitric oxide (NO) donor in the presence or absence of TEV48125, a fully humanized CGRP antibody. Cutaneous allodynia (CA) was used as an outcome measure and CGRP blood and cerebrospinal fluid (CSF) levels were measured. Results BLS and NO donor challenge evoked delayed, long-lasting CA selectively in rats that were previously treated with sumatriptan or morphine. BLS produced a significant increase in CGRP in the plasma, but not CSF, in animals that were previously exposed to sumatriptan compared to saline controls. TEV48125 did not modify baseline tactile thresholds or produce behavioral side effects, but significantly inhibited both BLS- and NO donor-induced CA in animals that were previously primed with sumatriptan or morphine; an isotype control protein that does not bind CGRP had no effect. Interpretation These data suggest that acute migraine medications may promote MOH in susceptible individuals through CGRP-dependent mechanisms and that anti-CGRP antibodies may be a useful clinical strategy for the treatment of MOH.

Kappa opioid receptor antagonists: A possible new class of therapeutics for migraine prevention.

Background Stress is the most commonly reported migraine trigger. Dynorphin, an endogenous opioid peptide acting preferentially at kappa opioid receptors (KORs), is a key mediator of stress responses. The aim of this study was to use an injury-free rat model of functional cephalic pain with features of migraine and medication overuse headache (MOH) to test the possible preventive benefit of KOR blockade on stress-induced cephalic pain. Methods Following sumatriptan priming to model MOH, rats were hyper-responsive to environmental stress, demonstrating delayed cephalic and extracephalic allodynia and increased levels of CGRP in the jugular blood, consistent with commonly observed clinical outcomes during migraine. Nor-binaltorphimine (nor-BNI), a long-acting KOR antagonist or CYM51317, a novel short-acting KOR antagonist, were given systemically either during sumatriptan priming or immediately before environmental stress challenge. The effects of KOR blockade in the amygdala on stress-induced allodynia was determined by administration of nor-BNI into the right or left central nucleus of the amygdala (CeA). Results KOR blockade prevented both stress-induced allodynia and increased plasma CGRP. Stress increased dynorphin content and phosphorylated KOR in both the left and right CeA in sumatriptan-primed rats. However, KOR blockade only in the right CeA prevented stress-induced cephalic allodynia as well as extracephalic allodynia, measured in either the right or left hindpaws. U69,593, a KOR agonist, given into the right, but not the left, CeA, produced allodynia selectively in sumatriptan-primed rats. Both stress and U69,593-induced allodynia were prevented by right CeA U0126, a mitogen-activated protein kinase inhibitor, presumably acting downstream of KOR. Conclusions Our data reveal a novel lateralized KOR circuit that mediated stress-induced cutaneous allodynia and increased plasma CGRP in an injury-free model of functional cephalic pain with features of migraine and medication overuse headache. Selective, small molecule, orally available, and reversible KOR antagonists are currently in development and may represent a novel class of preventive therapeutics for migraine.

Endogenous opioid activity in the anterior cingulate cortex is required for relief of pain.

Pain is aversive, and its relief elicits reward mediated by dopaminergic signaling in the nucleus accumbens (NAc), a part of the mesolimbic reward motivation pathway. How the reward pathway is engaged by pain-relieving treatments is not known. Endogenous opioid signaling in the anterior cingulate cortex (ACC), an area encoding pain aversiveness, contributes to pain modulation. We examined whether endogenous ACC opioid neurotransmission is required for relief of pain and subsequent downstream activation of NAc dopamine signaling. Conditioned place preference (CPP) and in vivo microdialysis were used to assess negative reinforcement and NAc dopaminergic transmission. In rats with postsurgical or neuropathic pain, blockade of opioid signaling in the rostral ACC (rACC) inhibited CPP and NAc dopamine release resulting from non-opioid pain-relieving treatments, including peripheral nerve block or spinal clonidine, an α2-adrenergic agonist. Conversely, pharmacological activation of rACC opioid receptors of injured, but not pain-free, animals was sufficient to stimulate dopamine release in the NAc and produce CPP. In neuropathic, but not sham-operated, rats, systemic doses of morphine that did not affect withdrawal thresholds elicited CPP and NAc dopamine release, effects that were prevented by blockade of ACC opioid receptors. The data provide a neural explanation for the preferential effects of opioids on pain affect and demonstrate that engagement of NAc dopaminergic transmission by non-opioid pain-relieving treatments depends on upstream ACC opioid circuits. Endogenous opioid signaling in the ACC appears to be both necessary and sufficient for relief of pain aversiveness.

Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states.

Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.

Biocompatible PEDOT:Nafion composite electrode coatings for selective detection of neurotransmitters in vivo.

A Nafion and poly(3,4-ethylenedioxythiophene) (PEDOT) containing composite polymer has been electropolymerized on carbon-fiber microelectrodes with the goal of creating a mechanically stable, robust, and controllable electrode coating that increases the selectivity and sensitivity of in vivo electrochemical measurements. The coating is deposited on carbon-fiber microelectrodes by applying a triangle waveform from +1.5 V to -0.8 V and back in a dilute solution of ethylenedioxythiophene (EDOT) and Nafion in acetonitrile. Scanning electron microscopy demonstrated that the coating is uniform and ∼100 nm thick. Energy-dispersive X-ray spectroscopy demonstrated that both sulfur and fluorine are present in the coating, indicating the incorporation of PEDOT (poly(3,4-ethylenedioxythiophene) and Nafion. Two types of PEDOT:Nafion coated electrodes were then analyzed electrochemically. PEDOT:Nafion-coated electrodes made using 200 µM EDOT exhibit a 10-90 response time of 0.46 ± 0.09 s versus 0.45 ± 0.11 s for an uncoated fiber in response to a 1.0 µM bolus of dopamine. The electrodes coated using a higher EDOT concentration (400 µM) are slower with a 10-90 response time of 0.84 ± 0.19 s, but display increased sensitivity to dopamine, at 46 ± 13 nA/µM, compared to 26 ± 6 nA/µM for the electrodes coated in 200 µM EDOT and 13 ± 2 nA/µM for an uncoated fiber. PEDOT:Nafion-coated electrodes were lowered into the nucleus accumbens of a rat, and both spontaneous and electrically evoked dopamine release were measured. In addition to improvements in sensitivity and selectivity, the coating dramatically reduces acute in vivo biofouling.

Pain relief produces negative reinforcement through activation of mesolimbic reward-valuation circuitry.

Relief of pain is rewarding. Using a model of experimental postsurgical pain we show that blockade of afferent input from the injury with local anesthetic elicits conditioned place preference, activates ventral tegmental dopaminergic cells, and increases dopamine release in the nucleus accumbens. Importantly, place preference is associated with increased activity in midbrain dopaminergic neurons and blocked by dopamine antagonists injected into the nucleus accumbens. The data directly support the hypothesis that relief of pain produces negative reinforcement through activation of the mesolimbic reward-valuation circuitry.

Discovery of amphipathic dynorphin A analogues to inhibit the neuroexcitatory effects of dynorphin A through bradykinin receptors in the spinal cord.

We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well-known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but it allowed us to discover a potential neuroexcitatory target. Well-known BR ligands, BK, [des-Arg(10), Leu(9)]-kallidin (DALKD), and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed, and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg(7)]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A's biological activity, is not necessary, and that a type I ß-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus, ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.

Low Risk of Clinically Important Traumatic Brain Injury in Children Who Tumble Down Stairs.

BACKGROUND: Pediatric trauma management seeks to minimize head computed tomography (HCT) while capturing clinically important traumatic brain injuries (ciTBI). The Pediatric Emergency Care Applied Research Network (PECARN) system stratifies patients as high-, intermediate-, or low-risk for ciTBI. Although designed for free falls, we noted that PECARN criteria often are applied to tumbling down stairs (TDS), with steps estimated at 12", though TDS rarely appeared to result in ciTBI. METHODS: In a retrospective chart review of pediatric TDS patients, data was collected on mechanism of injury, clinical presentation, imaging, and incidence of ciTBI. PECARN scores were developed under three models: TDS-12 (12″ steps), TDS-8 (more accurate 8" steps), and TDS-0 (TDS not a severe mechanism). RESULTS: 344 patients met criteria for study inclusion. Mean age was 6.3 years and 89 (26%) were <2 years. No patients had ciTBI. This included 88 patients who tumbled down 12 steps or more. Across all models, the same 7 patients (2.0%) were at high-risk for ciTBI. Intermediate- and low-risk cohorts were 287 (83%) and 50 (15%) for TDS-12, 171 (50%) and 166 (48%) for TDS-8, and 16 (4.7%) and 321 (93%) for TDS-0, respectively for each model. Under TDS-8, 116 (34%) patients shifted to the low-risk category. Under TDS-0, 271 (79%) patients shifted to the low-risk category, leaving only 23 patients (6.7%) at high- or intermediate-risk (n = 7, 16, respectively). CONCLUSIONS: In pediatric patients, the risk of ciTBI after TDS is low. TDS should not be treated as a free fall in risk assessment. TYPE OF STUDY: Retrospective Modeling Study. LEVEL OF EVIDENCE: Level III.

Potential mechanisms for osteopathic manipulative treatment to alleviate migraine-like pain in female rats.

Introduction: Migraines are the leading cause of disability in the United States, and the use of non-pharmaceutical treatments like osteopathic manipulative treatment (OMT) has shown promise. Despite its potential, the lack of mechanistic understanding has hindered widespread adoption. This study aims to investigate the efficacy of OMT in treating acute migraines and unravel its underlying mechanisms of action. Methods: Female rats were subjected to a "two-hit" approach to induce migraine-like pain. This involved bilateral injections of Complete Freund's Adjuvant (CFA) into the trapezius muscle (1st hit) followed by exposure to Umbellulone, a human migraine trigger, on Day 6 post-CFA (2nd hit). Soft tissue and articulatory techniques were applied to the cervical region for acute abortive or repeated prophylactic treatment. Cutaneous allodynia and trigeminal system activation were assessed through behavioral tests and immunohistochemical staining. Results: Following Umbellulone inhalation, CFA-primed rats exhibited periorbital and hind paw allodynia. Immediate application of OMT after Umbellulone inhalation as an abortive treatment partially alleviated cutaneous allodynia. With OMT applied thrice as a prophylactic measure, complete suppression of tactile hypersensitivity was observed. Prophylactic OMT also prevented the increase of c-fos signals in the trigeminal nucleus caudalis and the elevation of calcitonin gene-related peptide expression in trigeminal ganglia induced by CFA and Umbellulone exposure at 2 h post-inhalation. Discussion: These findings provide mechanistic insights into OMT's migraine-relief potential and underscore its viability as a non-pharmacological avenue for managing migraines.

CAP1 (cyclase-associated protein 1) mediates the cyclic AMP signals that activate Rap1 in stimulating matrix adhesion of colon cancer cells.

We previously reported that CAP1 (Cyclase-Associated Protein 1) regulates matrix adhesion in mammalian cells through FAK (Focal Adhesion Kinase). More recently, we discovered a phosphor-regulation mechanism for CAP1 through the Ser307/Ser309 tandem site that is of critical importance for all CAP1 functions. However, molecular mechanisms underlying the CAP1 function in adhesion and its regulation remain largely unknown. Here we report that Rap1 also facilitates the CAP1 function in adhesion, and more importantly, we identify a novel signaling pathway where CAP1 mediates the cAMP signals, through the cAMP effectors Epac (Exchange proteins directly activated by cAMP) and PKA (Protein Kinase A), to activate Rap1 in stimulating matrix adhesion in colon cancer cells. Knockdown of CAP1 led to opposite adhesion phenotypes in SW480 and HCT116 colon cancer cells, with reduced matrix adhesion and reduced FAK and Rap1 activities in SW480 cells while it stimulated matrix adhesion as well as FAK and Rap1 activities in HCT116 cells. Importantly, depletion of CAP1 abolished the stimulatory effects of the cAMP activators forskolin and isoproterenol, as well as that of Epac and PKA, on matrix adhesion in both cell types. Our results consistently support a required role for CAP1 in the cAMP activation of Rap1. Identification of the key role for CAP1 in linking the major second messenger cAMP to activation of Rap1 in stimulating adhesion, which may potentially also regulate proliferation in other cell types, not only vertically extends our knowledge on CAP biology, but also carries important translational potential for targeting CAP1 in cancer therapeutics.

Delving into the recent advancements of spinal cord injury treatment: a review of recent progress.

Spinal cord injury (SCI) research is a very complex field lending to why reviews of SCI literatures can be beneficial to current and future researchers. This review focuses on recent articles regarding potential modalities for the treatment and management of SCI. The modalities were broken down into four categories: neuroprotection-pharmacologic, neuroprotection-non-pharmacologic, neuroregeneration-pharmacologic, neuroregeneration-non-pharmacologic. Peer-reviewed articles were found using PubMed with search terms: "spinal cord injury", "spinal cord injury neuroregeneration", "olfactory ensheathing cells spinal cord injury", "rho-rock inhibitors spinal cord injury", "neural stem cell", "scaffold", "neural stem cell transplantation", "exosomes and SCI", "epidural stimulation SCI", "brain-computer interfaces and SCI". Most recent articles spanning two years were chosen for their relevance to the categories of SCI management and treatment. There has been a plethora of pre-clinical studies completed with their results being difficult to replicate in clinical studies. Therefore, scientists should focus on understanding and applying the results of previous research to develop more efficacious preclinical studies and clinical trials.

Letters to the editor: Nicotinic acetylcholine receptor ligands as potential targets for managing neuropathic pain induced by diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is a medical condition that is progressively becoming more prevalent. The underlying cause of DPN is still unknown, although there have been several hypothesized mechanisms. There are current pharmaceutical treatments used to manage the pain, but their efficacy is largely unsatisfactory and are often associated with serious adverse effects. This review will explore the evidence of a new potential target for treating DPN, the ligands for nicotinic acetylcholine receptors (nAChRs), specifically α4ꞵ2 agonists and α9α10 antagonists.

Gold Nanorod Substrate for Rat Fetal Neural Stem Cell Differentiation into Oligodendrocytes.

Gold nanorods (AuNRs) have been proposed to promote stem cell differentiation in vitro and in vivo. In this study, we examined a particular type of AuNR in supporting the differentiation of rat fetal neural stem cells (NSCs) into oligodendrocytes (ODCs). AuNRs were synthesized according to the seed-mediated method resulting in nanorods with an aspect ratio of around 3 (~12 nm diameter, 36 nm length) and plasmon resonance at 520 and 780 nm, as confirmed by transmission electron microscopy (TEM) and UV-vis spectroscopy, respectively. A layer-by-layer approach was used to fabricate the AuNR substrate on the functionalized glass coverslips. NSCs were propagated for 10 days using fibroblast growth factor, platelet-derived growth-factor-supplemented culture media, and differentiated on an AuNR or poly-D-lysine (PDL)-coated surface using differentiation media containing triiodothyronine for three weeks. Results showed that NSCs survived better and differentiated faster on the AuNRs compared to the PDL surface. By week 1, almost all cells had differentiated on the AuNR substrate, whereas only ~60% differentiated on the PDL surface, with similar percentages of ODCs and astrocytes. This study indicates that functionalized AuNR substrate does promote NSC differentiation and could be a viable tool for tissue engineering to support the differentiation of stem cells.

Functionalized Nanocellulose Drives Neural Stem Cells toward Neuronal Differentiation.

Transplantation of differentiated and fully functional neurons may be a better therapeutic option for the cure of neurodegenerative disorders and brain injuries than direct grafting of neural stem cells (NSCs) that are potentially tumorigenic. However, the differentiation of NSCs into a large population of neurons has been a challenge. Nanomaterials have been widely used as substrates to manipulate cell behavior due to their nano-size, excellent physicochemical properties, ease of synthesis, and versatility in surface functionalization. Nanomaterial-based scaffolds and synthetic polymers have been fabricated with topology resembling the micro-environment of the extracellular matrix. Nanocellulose materials are gaining attention because of their availability, biocompatibility, biodegradability and bioactivity, and affordable cost. We evaluated the role of nanocellulose with different linkage and surface features in promoting neuronal differentiation. Nanocellulose coupled with lysine molecules (CNC-Lys) provided positive charges that helped the cells to attach. Embryonic rat NSCs were differentiated on the CNC-Lys surface for up to three weeks. By the end of the three weeks of in vitro culture, 87% of the cells had attached to the CNC-Lys surface and more than half of the NSCs had differentiated into functional neurons, expressing endogenous glutamate, generating electrical activity and action potentials recorded by the multi-electrode array.

Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors.

Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male Sprague-Dawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80% of these cells co-express both receptors. Agonist-directed cell lesion in the RVM with the cytotoxin, saporin, using either CCK-saporin to target CCK receptor expressing cells, or dermorphin-saporin to target MOR expressing cells, resulted in concomitant loss of CCK2 and MOR expressing cells, did not alter the basal sensory thresholds but abolished the hyperalgesia induced by microinjection of CCK into the RVM. The findings suggest that these CCK2-MOR co-expressing RVM neurons facilitate pain and can be directly activated by CCK input to the RVM. Furthermore, lesion of these RVM neurons did not affect the initial development of neuropathic pain in the hind paw upon injury to the sciatic nerve, but the abnormal pain states were short lived such that by about day 9 the sensory thresholds had reverted to pre-injury baselines despite the existing neuropathy. These data support our hypothesis and identify CCK2-MOR co-expressing neurons in the RVM as potential therapeutic targets for neuropathic pain.