Chronic Morphine Modulates PDGFR-ß and PDGF-B Expression and Distribution in Dorsal Root Ganglia and Spinal Cord in Male Rats.

The analgesic effect of opioids decreases over time due to the development of analgesic tolerance. We have shown that inhibition of the platelet-derived growth factor beta (PDGFR-ß) signaling eliminates morphine analgesic tolerance in rats. Although the PDGFR-ß and its ligand, the platelet-derived growth factor type B (PDGF-B), are expressed in the substantia gelatinosa of the spinal cord (SG) and in the dorsal root ganglia (DRG), their precise distribution within different cell types of these structures is unknown. Additionally, the impact of a tolerance-mediating chronic morphine treatment, on the expression and distribution of PDGF-B and PDGFR-ß has not yet been studied. Using immunohistochemistry (IHC), we found that in the spinal cord, PDGFR-ß and PDGF-B were expressed in neurons and oligodendrocytes and co-localized with the mu-opioid receptor (MOPr) in opioid naïve rats. PDGF-B was also found in microglia and astrocytes. Both PDGFR-ß and PDGF-B were detected in DRG neurons but not in spinal primary afferent terminals. Chronic morphine exposure did not change the cellular distribution of PDGFR-ß or PDGF-B. However, PDGFR-ß expression was downregulated in the SG and upregulated in the DRG. Consistent with our previous finding that morphine caused tolerance by inducing PDGF-B release, PDGF-B was upregulated in the spinal cord. We also found that chronic morphine exposure caused a spinal proliferation of oligodendrocytes. The changes in PDGFR-ß and PDGF-B expression induced by chronic morphine treatment suggest potential mechanistic substrates underlying opioid tolerance.

Preventing spread of aerosolized infectious particles during medical procedures: A lab-based analysis of an inexpensive plastic enclosure.

Severe viral respiratory diseases, such as SARS-CoV-2, are transmitted through aerosol particles produced by coughing, talking, and breathing. Medical procedures including tracheal intubation, extubation, dental work, and any procedure involving close contact with a patient's airways can increase exposure to infectious aerosol particles. This presents a significant risk for viral exposure of nearby healthcare workers during and following patient care. Previous studies have examined the effectiveness of plastic enclosures for trapping aerosol particles and protecting health-care workers. However, many of these enclosures are expensive or are burdensome for healthcare workers to work with. In this study, a low-cost plastic enclosure was designed to reduce aerosol spread and viral transmission during medical procedures, while also alleviating issues found in the design and use of other medical enclosures to contain aerosols. This enclosure is fabricated from clear polycarbonate for maximum visibility. A large single-side cutout provides health care providers with ease of access to the patient with a separate cutout for equipment access. A survey of medical providers in a local hospital network demonstrated their approval of the enclosure's ease of use and design. The enclosure with appropriate plastic covers reduced total escaped particle number concentrations (diameter > 0.01 µm) by over 93% at 8 cm away from all openings. Concentration decay experiments indicated that the enclosure without active suction should be left on the patient for 15-20 minutes following a tracheal manipulation to allow sufficient time for >90% of aerosol particles to settle upon interior surfaces. This decreases to 5 minutes when 30 LPM suction is applied. This enclosure is an inexpensive, easily implemented additional layer of protection that can be used to help contain infectious or otherwise potentially hazardous aerosol particles while providing access into the enclosure.

Subanalgesic morphine doses augment fentanyl analgesia by interacting with delta opioid receptors in male rats.

Opioids are commonly used for the treatment of postoperative and post-traumatic pain; however, their therapeutic effectiveness is limited by undesirable and life-threatening side effects. Researchers have long attempted to develop opioid co-administration therapies that enhance analgesia, but the complexity of opioid analgesia and our incomplete mechanistic understanding has made this a daunting task. We discovered that subanalgesic morphine doses (100 ng/kg-10 µg/kg) augmented the acute analgesic effect of fentanyl (20 µg/kg) following subcutaneous drug co-administration to male rats. In addition, administration of equivalent drug ratios to naïve rat spinal cord membranes induced a twofold increase in G protein activation. The rate of GTP hydrolysis remained unchanged. We demonstrated that these behavioral and biochemical effects were mediated by the delta opioid receptor (DOP). Subanalgesic doses of the DOP-selective agonist SNC80 also augmented the acute analgesic effect of fentanyl. Furthermore, co-administration of the DOP antagonist naltrindole with both fentanyl-morphine and fentanyl-SNC80 combinations prevented augmentation of both analgesia and G protein activation. The mu opioid receptor (MOP) antagonist cyprodime did not block augmentation. Confocal microscopy of the substantia gelatinosa of rats treated with fentanyl, subanalgesic morphine, or this combination showed that changes in MOP internalization did not account for augmentation effects. Together, these findings suggest that augmentation of fentanyl analgesia by subanalgesic morphine is mediated by increased G protein activation resulting from a synergistic interaction between or heterodimerization of MOPs and DOPs. This finding is of great therapeutic significance because it suggests a strategy for the development of DOP-selective ligands that can enhance the therapeutic index of clinically used MOP drugs.

Artificial hyperventilation normalizes haemodynamics and arterial oxygen content in hypoxic rats.

INTRODUCTION: Although humans are capable of enduring critically low levels of oxygen, many hypoxaemic patients die despite aggressive therapies. Mimicking the physiological hyperventilation necessary to survive extreme hypoxic conditions could minimize the derangements caused by acute hypoxic-hypoxia. The objective of this study was to measure the haemodynamic-biochemical response to artificially induced hyperventilation in hypoxic rats. MATERIAL AND METHODS: Twenty-four deeply anaesthetized and mechanically ventilated rats were allocated to 3 groups: control (n = 5, FiO2 = 1); hypoxic spontaneously hyperventilating (n = 10, FiO2 = 0.08); and hypoxic artificially induced hyperventilation (n = 9, targeting PaCO2 = 10 mm Hg, FiO2 = 0.08). We compared the spontaneously and artificially hyperventilating groups. P-values < 0.01 were considered statistically significant. Mean arterial pressure (MAP) and serum chemistry were measured for 180 minutes. RESULTS: The control group remained stable throughout the experiment. The hypoxic groups developed profound hypotension after the decrease in FiO2. However, the artificially induced hyperventilated rats recovered their MAP to levels higher than the spontaneously hyperventilating group (117.1 ± 17.2 vs. 68.1 ± 16.0, P = 0.0048). In regard to the biochemical derangements, even though the serum lactate and PaO2 were not different among the hypoxic groups, the artificially hyperventilated group achieved significantly higher SaO2 (94.3 ± 3.6 vs. 58.6 ± 9.6, P = 0.005), pH (7.87 ± 0.04 vs. 7.50 ± 0.13, P = 0.005), and CaO2 (17.7 ± 2.6 vs. 10.2 ± 1.3, P = 0.005) at 180 minutes. CONCLUSIONS: Artificially induced hyperventilation led to the correction of arterial oxygen content, severe serum chemistry, and haemodynamic derangements. These findings may represent a novel rescue manoeuvre and serve as a bridge to a permanent form of support, but should be further studied before being translated to the clinical setting.

Use of healthcare resources in patients with low back pain and comorbid depression or anxiety.

BACKGROUND CONTEXT: Psychological comorbidities are important prognostic factors for low back pain (LBP). To develop improved treatment paradigms, it is first necessary to characterize and determine current patterns of treatment in this population. PURPOSE: Identify how comorbid depression or anxiety in patients with LBP is related to use of healthcare resources. STUDY DESIGN/SETTING: Retrospective cohort study using electronic health records from outpatient offices at a large multisite academic medical center. PATIENT SAMPLE: Data from 513,088 unique patients seen between January 2010 and July 2020 (58.0% female, 52.6±19.5 years) with a diagnosis of LBP, indicated by predetermined ICD-9 and ICD-10 codes. OUTCOME MEASURES: Average self-reported pain scores, absolute differences and unadjusted risk ratios to compare opioid use, emergency department visits, hospitalizations, advanced imaging orders, spinal injections, and back surgeries between cohorts. METHODS: Clinical characteristics and data regarding use of healthcare resources were extracted from the electronic health record. Clinical features and patterns in healthcare utilization were determined for patients with depression or anxiety compared to those without. RESULTS: Depression or anxiety was coded for 21.4% of patients at first LBP visit. Those with depression or anxiety were more likely to be on opioids (unadjusted risk ratio: 1.22, CI: [1.22,1.23]), go to the emergency department (1.31 [1.30-1.33]), be hospitalized (1.15 [1.13, 1.17]), receive advanced imaging (1.09 [1.08, 1.11]), receive an epidural steroid injection (1.16 [1.15, 1.18]), and less likely to have back surgery (0.74 [0.72, 0.77]). Differences in pain scores for those with depression/anxiety compared to those without were not clinically significant. CONCLUSIONS: Depression/anxiety is associated with increased use of healthcare resources, and is not associated with clinically meaningful elevated pain scores. Limitations come from use of an aggregate data set and reliance on administrative coding.

Neuropathic pain generates silent synapses in thalamic projection to anterior cingulate cortex.

ABSTRACT: Pain experience can change the central processing of nociceptive inputs, resulting in persistent allodynia and hyperalgesia. However, the underlying circuit mechanisms remain underexplored. Here, we focus on pain-induced remodeling of the projection from the mediodorsal thalamus (MD) to the anterior cingulate cortex (ACC), a projection that relays spinal nociceptive input for central processing. Using optogenetics combined with slice electrophysiology, we detected in male mice that 7 days of chronic constriction injury (CCI; achieved by loose ligation of the sciatic nerve) generated AMPA receptor (AMPAR)-silent glutamatergic synapses within the contralateral MD-to-ACC projection. AMPAR-silent synapses are typically GluN2B-enriched nascent glutamatergic synapses that mediate the initial formation of neural circuits during early development. During development, some silent synapses mature and become "unsilenced" by recruiting and stabilizing AMPARs, consolidating and strengthening the newly formed circuits. Consistent with these synaptogenic features, pain-induced generation of silent synapses was accompanied by increased densities of immature dendritic spines in ACC neurons and increased synaptic weight of GluN2B-containing NMDA receptors (NMDARs) in the MD-to-ACC projection. After prolonged (∼30 days) CCI, injury-generated silent synapses declined to low levels, which likely resulted from a synaptic maturation process that strengthens AMPAR-mediated MD-to-ACC transmission. Consistent with this hypothesis, viral-mediated knockdown of GluN2B in ACC neurons, which prevented pain-induced generation of silent synapses and silent synapse-mediated strengthening of MD-to-ACC projection after prolonged CCI, prevented the development of allodynia. Taken together, our results depict a silent synapse-mediated mechanism through which key supraspinal neural circuits that regulate pain sensitivity are remodeled to induce allodynia and hyperalgesia.

EGFR Signaling Causes Morphine Tolerance and Mechanical Sensitization in Rats.

The safety and efficacy of opioids are compromised as analgesic tolerance develops. Opioids are also ineffective against neuropathic pain. Recent reports have suggested that inhibitors of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), may have analgesic effects in cancer patients suffering from neuropathic pain. It has been shown that the platelet-derived growth factor receptor-ß (PDGFR-ß), an RTK that has been shown to interact with the EGFR, mediates opioid tolerance but does not induce analgesia. Therefore, we sought to determine whether EGFR signaling was involved in opioid tolerance and whether EGFR and PDGFR signaling could induce pain in rats. We found that gefitinib, an EGFR antagonist, eliminated morphine tolerance. In addition, repeated EGF administration rendered animals unresponsive to subsequent analgesic doses of morphine, a phenomenon we call "pre-tolerance." Using a nerve injury model, we found that gefitinib alone was not analgesic. Rather, it reversed insensitivity to morphine analgesia (pre-tolerance) caused by the release of EGF by injured nerves. We also showed that repeated, but not acute EGF or PDGF-BB administration induced mechanical hypersensitivity in rats. EGFR and PDGFR-ß signaling interacted to produce this sensitization. EGFR was widely expressed in primary sensory afferent cell bodies, demonstrating a neuroanatomical substrate for our findings. Taken together, our results suggest a direct mechanistic link between opioid tolerance and mechanical sensitization. EGFR antagonism could eventually play an important clinical role in the treatment of opioid tolerance and neuropathic pain that is refractory to opioid treatment.

An assay for chemical nociception in Drosophila larvae.

Chemically induced nociception has not yet been studied intensively in genetically tractable models. Hence, our goal was to establish a Drosophila assay that can be used to study the cellular and molecular/genetic bases of chemically induced nociception. Drosophila larvae exposed to increasing concentrations of hydrochloric acid (HCl) produced an increasingly intense aversive rolling response. HCl (0.5%) was subthreshold and provoked no response. All classes of peripheral multidendritic (md) sensory neurons (classes I-IV) are required for full responsiveness to acid, with class IV making the largest contribution. At the cellular level, classes IV, III and I showed increases in calcium following acid exposure. In the central nervous system, Basin-4 second-order neurons are the key regulators of chemically induced nociception, with a slight contribution from other types. Finally, chemical nociception can be sensitized by tissue damage. Subthreshold HCl provoked chemical allodynia in larvae 4 h after physical puncture wounding. Pinch wounding and UV irradiation, which do not compromise the cuticle, did not cause chemical allodynia. In sum, we developed a novel assay to study chemically induced nociception in Drosophila larvae. This assay, combined with the high genetic resolving power of Drosophila, should improve our basic understanding of fundamental mechanisms of chemical nociception. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Growth Factor Signaling Regulates Mechanical Nociception in Flies and Vertebrates.

Mechanical sensitization is one of the most difficult clinical pain problems to treat. However, the molecular and genetic bases of mechanical nociception are unclear. Here we develop a Drosophila model of mechanical nociception to investigate the ion channels and signaling pathways that regulate mechanical nociception. We fabricated von Frey filaments that span the subthreshold to high noxious range for Drosophila larvae. Using these, we discovered that pressure (force/area), rather than force per se, is the main determinant of aversive rolling responses to noxious mechanical stimuli. We demonstrated that the RTK PDGF/VEGF receptor (Pvr) and its ligands (Pvfs 2 and 3) are required for mechanical nociception and normal dendritic branching. Pvr is expressed and functions in class IV sensory neurons, whereas Pvf2 and Pvf3 are produced by multiple tissues. Constitutive overexpression of Pvr and its ligands or inducible overexpression of Pvr led to mechanical hypersensitivity that could be partially separated from morphological effects. Genetic analyses revealed that the Piezo and Pain ion channels are required for mechanical hypersensitivity observed upon ectopic activation of Pvr signaling. PDGF, but not VEGF, peptides caused mechanical hypersensitivity in rats. Pharmacological inhibition of VEGF receptor Type 2 (VEGFR-2) signaling attenuated mechanical nociception in rats, suggesting a conserved role for PDGF and VEGFR-2 signaling in regulating mechanical nociception. VEGFR-2 inhibition also attenuated morphine analgesic tolerance in rats. Our results reveal that a conserved RTK signaling pathway regulates baseline mechanical nociception in flies and rats.SIGNIFICANCE STATEMENT Hypersensitivity to touch is poorly understood and extremely difficult to treat. Using a refined Drosophila model of mechanical nociception, we discovered a conserved VEGF-related receptor tyrosine kinase signaling pathway that regulates mechanical nociception in flies. Importantly, pharmacological inhibition of VEGF receptor Type 2 signaling in rats causes analgesia and blocks opioid tolerance. We have thus established a robust, genetically tractable system for the rapid identification and functional analysis of conserved genes underlying mechanical pain sensitivity.

Platelet-derived growth factor activates nociceptive neurons by inhibiting M-current and contributes to inflammatory pain.

Endogenous inflammatory mediators contribute to the pathogenesis of pain by acting on nociceptors, specialized sensory neurons that detect noxious stimuli. Here, we describe a new factor mediating inflammatory pain. We show that platelet-derived growth factor (PDGF)-BB applied in vitro causes repetitive firing of dissociated nociceptor-like rat dorsal root ganglion neurons and decreased their threshold for action potential generation. Injection of PDGF-BB into the paw produced nocifensive behavior in rats and led to thermal and mechanical pain hypersensitivity. We further detailed the biophysical mechanisms of these PDGF-BB effects and show that PDGF receptor-induced inhibition of nociceptive M-current underlies PDGF-BB-mediated nociceptive hyperexcitability. Moreover, in vivo sequestration of PDGF or inhibition of the PDGF receptor attenuates acute formalin-induced inflammatory pain. Our discovery of a new pain-facilitating proinflammatory mediator, which by inhibiting M-current activates nociceptive neurons and thus contributes to inflammatory pain, improves our understanding of inflammatory pain pathophysiology and may have important clinical implications for pain treatment.