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.

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.

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.

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.

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.

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.

Predominant differentiation of rat fetal neural stem cells into functional oligodendrocytes in vitro.

Oligodendrocytes form myelin in the CNS. A fast method to produce large quantity of oligodendrocytes that have the capacity of myelinating CNS neurons would be very useful for treating CNS injuries or demyelinating diseases, or for research purposes. We developed a simple standard protocol for predominant differentiation of rat fetal neural stem cells (NSCs) into oligodendrocytes. We adopted a new method to purify the oligodendrocytes and co-cultured the newly differentiated oligodendrocytes with hippocampal neurons to confirm their myelination capability. NSCs from embryonic day 14 (E14) were propagated at the presence of basic fibroblast growth factor and platelet derived growth factor alpha, and then differentiated in the medium containing triiodothyronine. Four extracellular matrix (ECM), poly-d-lysine (PDL), PDL-laminin, fibronectin, and matrigel, were examined for NSC differentiation. About 90 % of NSCs differentiated into oligodendrocytes on matrigel compared to 32 % on PDL or PDL-laminin, and 26 % on fibronectin after 3 weeks of differentiation, demonstrating the significant influence of ECM. Further, newly differentiated oligodendrocytes were co-cultured with hippocampal neurons from E18 rat embryos resulting in robust myelination of neurites at three weeks. In summary, we present a simplified and efficient method to predominantly generate oligodendrocytes from NSCs that is potentially very useful for CNS demyelination diseases.

Glioma-derived exosomes drive the differentiation of neural stem cells to astrocytes.

Exosomes appear to be effective inter-cellular communicators delivering several types of molecules, such as proteins and RNAs, suggesting that they could influence neural stem cell (NSC) differentiation. Our RNA sequencing studies demonstrated that the RNAs related to cell proliferation and astrocyte differentiation were upregulated in human mesenchymal stem cells (hMSC) when co-cultured with exosomes obtained from the culture medium of human glioma cells (U87). Metallothionein 3 and elastin genes, which are related to cell proliferation, increased 10 and 7.2 fold, respectively. Expression of genes for astrocyte differentiation, such as tumor growth factor alpha, induced protein 3 of the NOTCH1 family, colony stimulating factor and interleukin 6 of the STAT3 family and Hes family bHLH transcription factor 1 also increased by 2.3, 10, 4.7 and 2.9 fold, respectively. We further examined the effects of these exosomes on rat fetal neural stem cell (rNSC) differentiation using the secreted exosomes from U87 glioma cells or exosomes from U87 cells that were stimulated with interleukin 1ß (IL-1ß). The rNSCs, extracted from rat brains at embryonic day 14 (E14), underwent a culture protocol that normally leads to predominant (~90%) differentiation to ODCs. However, in the presence of the exosomes from untreated or IL-1ß-treated U87 cells, significantly more cells differentiated into astrocytes, especially in the presence of exosomes obtained from the IL-1ß-challenged glioma cells. Moreover, glioma-derived exosomes appeared to inhibit rNSC differentiation into ODCs or astrocytes as indicated by a significantly increased population of unlabeled cells. A portion of the resulting astrocytes co-expressed both CD133 and glial fibrillary acidic protein (GFAP) suggesting that exosomes from U87 cells could promote astrocytic differentiation of NSCs with features expected from a transformed cell. Our data clearly demonstrated that exosomes secreted by human glioma cells provide a strong driving force for rat neural stem cells to differentiate into astrocytes, uncovering potential pathways and therapeutic targets that might control this aggressive tumor type.

Multiple sites and actions of gabapentin-induced relief of ongoing experimental neuropathic pain.

Gabapentin (GBP) is a first-line therapy for neuropathic pain, but its mechanisms and sites of action remain uncertain. We investigated GBP-induced modulation of neuropathic pain following spinal nerve ligation (SNL) in rats. Intravenous or intrathecal GBP reversed evoked mechanical hypersensitivity and produced conditioned place preference (CPP) and dopamine (DA) release in the nucleus accumbens (NAc) selectively in SNL rats. Spinal GBP also significantly inhibited dorsal horn wide-dynamic-range neuronal responses to a range of evoked stimuli in SNL rats. By contrast, GBP microinjected bilaterally into the rostral anterior cingulate cortex (rACC), produced CPP, and elicited NAc DA release selectively in SNL rats but did not reverse tactile allodynia and had marginal effects on wide-dynamic-range neuronal activity. Moreover, blockade of endogenous opioid signaling in the rACC prevented intravenous GBP-induced CPP and NAc DA release but failed to block its inhibition of tactile allodynia. Gabapentin, therefore, can potentially act to produce its pain relieving effects by (a) inhibition of injury-induced spinal neuronal excitability, evoked hypersensitivity, and ongoing pain and (b) selective supraspinal modulation of affective qualities of pain, without alteration of reflexive behaviors. Consistent with previous findings of pain relief from nonopioid analgesics, GBP requires engagement of rACC endogenous opioid circuits and downstream activation of mesolimbic reward circuits reflected in learned pain-motivated behaviors. These findings support the partial separation of sensory and affective dimensions of pain in this experimental model and suggest that modulation of affective-motivational qualities of pain may be the preferential mechanism of GBP's analgesic effects in patients.

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.

Discovery of Stable Non-opioid Dynorphin A Analogues Interacting at the Bradykinin Receptors for the Treatment of Neuropathic Pain.

Dynorphin A (Dyn A) is a unique endogenous ligand that possesses well-known neuroinhibitory effects via opioid receptors with a preference for the kappa receptor but also neuroexcitatory effects, which cause hyperalgesia. We have shown that the neuroexcitatory effects are mediated through bradykinin (BK) receptors and that intrathecal (i.th.) administration of our lead ligand 1, [des-Arg7]-Dyn A-(4-11), which shows good binding affinity (IC50 = 150 nM) at the BK receptors, blocks Dyn A-induced hyperalgesia in naïve animals and reverses thermal and tactile hypersensitivities in a dose-dependent manner in nerve-injured animals. However, 1 has a serious drawback as a potential drug candidate for the treatment of neuropathic pain because of its susceptibility to enzymatic degradation. In an effort to increase its stability, we modified ligand 1 using non-natural amino acids and found that analogues substituted at or near the N-terminus with a d-isomer retain binding at the receptor and provide a large increase in stability. In particular when Leu5 was modified, with either the d-isomer or N-methylation, there was a large increase in stability (t1/2 = 0.7-160 h in rat plasma) observed. From these studies, we have developed a very stable Dyn A analogue 16, [d-Leu5,des-Arg7]-Dyn A-(4-11), that binds to BK receptors (IC50 = 130 nM) in the same range as ligand 1 and shows good antihyperalgesic effects in both naïve rats and L5/L6 spinal nerve ligation rats.

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.

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.

Evaluation of reward from pain relief.

The human experience of pain is multidimensional and comprises sensory, affective, and cognitive dimensions. Preclinical assessment of pain has been largely focused on the sensory features that contribute to nociception. The affective (aversive) qualities of pain are clinically significant but have received relatively less mechanistic investigation in preclinical models. Recently, operant behaviors such as conditioned place preference, avoidance, escape from noxious stimulus, and analgesic drug self-administration have been used in rodents to evaluate affective aspects of pain. An important advance of such operant behaviors is that these approaches may allow the detection and mechanistic investigation of spontaneous neuropathic or ongoing inflammatory/nociceptive (i.e., nonevoked) pain that is otherwise difficult to assess in nonverbal animals. Operant measures may allow the identification of mechanisms that contribute differentially to reflexive hypersensitivity or to pain affect and may inform the decision to progress novel mechanisms to clinical trials for pain therapy. Additionally, operant behaviors may allow investigation of the poorly understood mechanisms and neural circuits underlying motivational aspects of pain and the reward of pain relief.