Selective targeting of peripheral cannabinoid receptors prevents behavioral symptoms and sensitization of trigeminal neurons in mouse models of migraine and medication overuse headache.

ABSTRACT: Migraine affects ∼15% of the world's population greatly diminishing their quality of life. Current preventative treatments are effective in only a subset of migraine patients, and although cannabinoids seem beneficial in alleviating migraine symptoms, central nervous system side effects limit their widespread use. We developed peripherally restricted cannabinoids (PRCBs) that relieve chronic pain symptoms of cancer and neuropathies, without appreciable central nervous system side effects or tolerance development. Here, we determined PRCB effectiveness in alleviating hypersensitivity symptoms in mouse models of migraine and medication overuse headache. Long-term glyceryl trinitrate (GTN, 10 mg/kg) administration led to increased sensitivity to mechanical stimuli and increased expression of phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor potential ankyrin 1 proteins in trigeminal ganglia. Peripherally restricted cannabinoid pretreatment, but not posttreatment, prevented behavioral and biochemical correlates of GTN-induced sensitization. Low pH-activated and allyl isothiocyanate-activated currents in acutely isolated trigeminal neurons were reversibly attenuated by PRCB application. Long-term GTN treatment significantly enhanced these currents. Long-term sumatriptan treatment also led to the development of allodynia to mechanical and cold stimuli that was slowly reversible after sumatriptan discontinuation. Subsequent challenge with a previously ineffective low-dose GTN (0.1-0.3 mg/kg) revealed latent behavioral sensitization and increased expression of phosphorylated protein kinase A, neuronal nitric oxide synthase, and transient receptor potential ankyrin 1 proteins in trigeminal ganglia. Peripherally restricted cannabinoid pretreatment prevented all behavioral and biochemical correlates of allodynia and latent sensitization. Importantly, long-term PRCB treatment alone did not produce any behavioral or biochemical signs of sensitization. These data validate peripheral cannabinoid receptors as potential therapeutic targets in migraine and medication overuse headache.

Molecular consequences of fetal alcohol exposure on amniotic exosomal miRNAs with functional implications for stem cell potency and differentiation.

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH's effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.

A Role for The P2Y1 Receptor in Nonsynaptic Cross-depolarization in the Rat Dorsal Root Ganglia.

Non-synaptic transmission is pervasive throughout the nervous system. It appears especially prevalent in peripheral ganglia, where non-synaptic interactions between neighboring cell bodies have been described in both physiological and pathological conditions, a phenomenon referred to as cross-depolarization (CD) and thought to play a role in sensory processing and chronic pain. CD has been proposed to be mediated by a chemical agent, but its identity has remained elusive. Here, we report that in the rat dorsal root ganglion (DRG), the P2Y1 purinergic receptor (P2RY1) plays an important role in regulating CD. The effect of P2RY1 is cell-type specific: pharmacological blockade of P2RY1 inhibited CD in A-type neurons while enhancing it in C-type neurons. In the nodose ganglion of the vagus, CD requires extracellular calcium in a large percentage of cells. In contrast, we show that in the DRG extracellular calcium appears to play no major role, pointing to a mechanistic difference between the two peripheral ganglia. Furthermore, we show that DRG glial cells also play a cell-type specific role in CD regulation. Fluorocitrate-induced glial inactivation had no effect on A-cells but enhanced CD in C-cells. These findings shed light on the mechanism of CD in the DRG and pave the way for further analysis of non-synaptic neuronal communication in sensory ganglia.

Synthetic peripherally-restricted cannabinoid suppresses chemotherapy-induced peripheral neuropathy pain symptoms by CB1 receptor activation.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and dose-limiting side effect of cancer treatment that affects millions of cancer survivors throughout the world and current treatment options are extremely limited by their side effects. Cannabinoids are highly effective in suppressing pain symptoms of chemotherapy-induced and other peripheral neuropathies but their widespread use is limited by central nervous system (CNS)-mediated side effects. Here, we tested one compound from a series of recently developed synthetic peripherally restricted cannabinoids (PRCBs) in a rat model of cisplatin-induced peripheral neuropathy. Results show that local or systemic administration of 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI) dose-dependently suppressed CIPN mechanical and cold allodynia. Orally administered PrNMI also dose-dependently suppressed CIPN allodynia symptoms in both male and female rats without any CNS side effects. Co-administration with selective cannabinoid receptor subtype blockers revealed that PrNMI's anti-allodynic effects are mediated by CB1 receptor (CB1R) activation. Expression of CB2Rs was reduced in dorsal root ganglia from CIPN rats, whereas expression of CB1Rs and various endocannabinoid synthesizing and metabolizing enzymes was unaffected. Daily PrNMI treatment of CIPN rats for two weeks showed a lack of appreciable tolerance to PrNMI's anti-allodynic effects. In an operant task which reflects cerebral processing of pain, PrNMI also dose-dependently suppressed CIPN pain behaviors. Our results demonstrate that PRCBs exemplified by PrNMI may represent a viable option for the treatment of CIPN pain symptoms.

Peripherally restricted cannabinoid 1 receptor agonist as a novel analgesic in cancer-induced bone pain.

Many malignant cancers, including breast cancer, have a propensity to invade bones, leading to excruciating bone pain. Opioids are the primary analgesics used to alleviate this cancer-induced bone pain (CIBP) but are associated with numerous severe side effects, including enhanced bone degradation, which significantly impairs patients' quality of life. By contrast, agonists activating only peripheral CB1 receptors (CB1Rs) have been shown to effectively alleviate multiple chronic pain conditions with limited side effects, yet no studies have evaluated their role(s) in CIBP. Here, we demonstrate for the first time that a peripherally selective CB1R agonist can effectively suppress CIBP. Our studies using a syngeneic murine model of CIBP show that both acute and sustained administration of a peripherally restricted CB1R agonist, 4-{2-[-(1E)-1[(4-propylnaphthalen-1-yl)methylidene]-1H-inden-3-yl]ethyl}morpholine (PrNMI), significantly alleviated spontaneous pain behaviors in the animals. This analgesic effect by PrNMI can be reversed by a systemic administration but not spinal injection of SR141716, a selective CB1R antagonist. In addition, the cancer-induced bone loss in the animals was not exacerbated by a repeated administration of PrNMI. Furthermore, catalepsy and hypothermia, the common side effects induced by cannabinoids, were measured at the supratherapeutic doses of PrNMI tested. PrNMI induced mild sedation, yet no anxiety or a decrease in limb movements was detected. Overall, our studies demonstrate that CIBP can be effectively managed by using a peripherally restricted CB1R agonist, PrNMI, without inducing dose-limiting central side effects. Thus, targeting peripheral CB1Rs could be an alternative therapeutic strategy for the treatment of CIBP.

Relationship of axonal voltage-gated sodium channel 1.8 (NaV1.8) mRNA accumulation to sciatic nerve injury-induced painful neuropathy in rats.

Painful peripheral neuropathy is a significant clinical problem; however, its pathological mechanism and effective treatments remain elusive. Increased peripheral expression of tetrodotoxin-resistant voltage-gated sodium channel 1.8 (NaV1.8) has been shown to associate with chronic pain symptoms in humans and experimental animals. Sciatic nerve entrapment (SNE) injury was used to develop neuropathic pain symptoms in rats, resulting in increased NaV1.8 mRNA in the injured nerve but not in dorsal root ganglia (DRG). To study the role of NaV1.8 mRNA in the pathogenesis of SNE-induced painful neuropathy, NaV1.8 shRNA vector was delivered by subcutaneous injection of cationized gelatin/plasmid DNA polyplex into the rat hindpaw and its subsequent retrograde transport via sciatic nerve to DRG. This in vivo NaV1.8 shRNA treatment reversibly and repeatedly attenuated the SNE-induced pain symptoms, an effect that became apparent following a distinct lag period of 3-4 days and lasted for 4-6 days before returning to pretreatment levels. Surprisingly, apparent knockdown of NaV1.8 mRNA occurred only in the injured nerve, not in the DRG, during the pain alleviation period. Levels of heteronuclear NaV1.8 RNA were unaffected by SNE or shRNA treatments, suggesting that transcription of the Scn10a gene encoding NaV1.8 was unchanged. Based on these data, we postulate that increased axonal mRNA transport results in accumulation of functional NaV1.8 protein in the injured nerve and the development of painful neuropathy symptoms. Thus, targeted delivery of agents that interfere with axonal NaV1.8 mRNA may represent effective neuropathic pain treatments.

Altered ATP release and metabolism in dorsal root ganglia of neuropathic rats.

BACKGROUND: Adenosine 5'-triphosphate (ATP) has a ubiquitous role in metabolism and a major role in pain responses after tissue injury. We investigated the changes in basal and KCl-evoked ATP release from rat dorsal root ganglia (DRG) after peripheral neuropathy induction by unilateral sciatic nerve entrapment (SNE). RESULTS: After SNE, rats develop long-lasting decreases in ipsilateral hindpaw withdrawal thresholds to mechanical and thermal stimulation. At 15-21 days after neuropathy induction, excised ipsilateral L4-L5 DRG display significantly elevated basal extracellular ATP levels compared to contralateral or control (naive) DRG. However, KCl-evoked ATP release is no longer observed in ipsilateral DRG. We hypothesized that the differential SNE effects on basal and evoked ATP release could result from the conversion of extracellular ATP to adenosine with subsequent activation of adenosine A1 receptors (A1Rs) on DRG neurons. Adding the selective A1R agonist, 2-chloro-N(6)-cyclopentyladenosine (100 nM) significantly decreased basal and evoked ATP release in DRG from naïve rats, indicating functional A1R activation. In DRG ipsilateral to SNE, adding a selective A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (30 nM), further increased basal ATP levels and relieved the blockade of KCl-evoked ATP release suggesting that increased A1R activation attenuates evoked ATP release in neurons ipsilateral to SNE. To determine if altered ATP release was a consequence of altered DRG metabolism we compared O(2) consumption between control and neuropathic DRG. DRG ipsilateral to SNE consumed O(2) at a higher rate than control or contralateral DRG. CONCLUSION: These data suggest that peripheral nerve entrapment increases DRG metabolism and ATP release, which in turn is modulated by increased A1R activation.

Subcutaneous peripheral injection of cationized gelatin/DNA polyplexes as a platform for non-viral gene transfer to sensory neurons.

Selective modulation of sensory neuron gene expression could have numerous applications for the peripheral nervous system. Here, we report that subcutaneous peripheral injection of plasmid DNA complexed with a non-viral cationized gelatin (CG) vector led to transgene expression in rat lumbar dorsal root ganglia (DRGs). CG/DNA polyplexes appeared to undergo rapid retrograde transport through sciatic and spinal nerves, with reporter gene messenger RNA (mRNA) expression detectable in L4 and L5 DRGs within 60 hours. Maximum transgene expression was observed for polyplexes formed at 7.5:1 CG-to-DNA weight ratio under salt-free conditions, which generated 615 +/- 112 nm nanoparticles with zeta-potential of 9.4 +/- 0.19 mV. Six days after injection of the CG/DNA polypex, reporter gene protein immunofluorescence was observed in 1,164 +/- 176 DRG neurons, representing an estimated transfection rate of 47% of targeted neurons. Reporter gene expression was not detected in heart, lung, or liver tissues, suggesting a lack of systemic uptake. Measurements of tactile sensitivity indicate that CG/DNA injection did not cause behavioral toxicity. The injection platform was further used for plasmid-driven short hairpin RNA-mediated suppression of glyceraldehyde-3-phosphate dehydrogenase. This non-invasive gene delivery system could be used for the mechanistic study and targeted molecular evaluation of peripheral nervous system pathologies such as neuropathic pain.

Cell-permeant calcium buffer induced neuroprotection after cortical devascularization.

An excitotoxic cascade resulting in a significant intracellular calcium load is thought to be a primary mechanism leading to neuronal death after ischemia. One way to protect neurons from injury is through the use of cell-permeant calcium buffers. These molecules have been reported to be neuroprotective via their ability to increase the cell's overall Ca(2+) buffering load as well as by attenuating neurotransmitter release. However, their efficacy when given after injury has yet to be determined. We used diffusion-weighted magnetic resonance imaging (DWI), histological, and immunohistochemical methods to determine the neuroprotective efficacy of 2-aminophenol-N, N, O-triacetic acid acetoxymethyl ester (APTRA-AM) after focal cerebral ischemia. Injured animals were given two injections of APTRA-AM at 1 and 12 h after injury. Animals were imaged prior to injury and then at 12, 24, 48 h and 3 and 7 days after injury. After 7 days the animals were euthanized for correlative cresyl violet histology and immunohistochemistry. Injury resulted in a decrease in the apparent diffusion coefficient (ADC) of the injured area within the first 12 h of injury, which returned to normal by 7 days. In contrast, animals injected with APTRA-AM showed no significant change in the ADC at any time point studied. Tissue analysis showed that APTRA-AM significantly reduced the infarct size by 85% and extent of inflammatory cell infiltration by 94%. The results clearly demonstrate significant neuroprotection by APTRA-AM when given after injury.

Microdialysis in trigeminal ganglia.

Recent evidence demonstrates that neurons in sensory ganglia contribute to sensory signaling in both physiological and pathological states. In vivo sampling from this site may provide important insights into which substances mediate or modulate sensory transmission. To address this possibility, we have applied the microdialysis technique to the guinea pig trigeminal ganglia (TG). The large size and easy access of the TG in the guinea pig make it an ideal sampling site, while the somatotopic organization allows for specific regions of innervation to be studied at the ganglionic level. This report describes the use of microdialysis probes within the TG and use recovery and analysis of substance P (SP) and adenosine triphosphate (ATP) as case in points. Various physiological and pharmacological manipulations can be made, for example release of peptides from ganglionic neurons can be monitored in the presence or absence of inflammation in the orofacial region. Microdialysis performed in the TG thus provides a valuable site for recovery and measurement of a variety of extracellular substances that may be integral in the processing of trigeminal sensory information.