Synthesis of C-linked α-Gal and α-GalNAc-1'-hydroxyalkanes by way of C2 functionality transfer.

Inspired by reports of water sculpted Tn antigen (α-GalNAc-O-Ser/Thr) epitopes and our interest in producing metabolically more stable C-linked analogs of Tn, we explored the utility of C2 functionality on α-Gal-C-alkenes to deliver hydroxyl to the pendant alkenyl chain. Toward this end, a cyclic carbonate approach gave rise to a single C-linked α-Gal-1'(S)-hydroxyethane in 3 steps, and use of a 2-(hydroxyimino)galactoside cyclization transferred an oxygen to a pendant cis-substituted C-linked alkene affording the R-configuration at the newly formed stereocenter (7:1 dr). Reduction and acetylation of the resultant isoxazoline demonstrated this approach as a viable route to C-linked α-GalNAc-1'-hydroxyalkanes.

Transcriptional Changes in Dorsal Spinal Cord Persist after Surgical Incision Despite Preemptive Analgesia with Peripheral Resiniferatoxin.

BACKGROUND: Peripheral nociceptors expressing the ion channel transient receptor potential cation channel, subfamily V, member 1, play an important role in mediating postoperative pain. Signaling from these nociceptors in the peri- and postoperative period can lead to plastic changes in the spinal cord and, when controlled, can yield analgesia. The transcriptomic changes in the dorsal spinal cord after surgery, and potential coupling to transient receptor potential cation channel, subfamily V, member 1-positive nociceptor signaling, remain poorly studied. METHODS: Resiniferatoxin was injected subcutaneously into rat hind paw several minutes before surgical incision to inactivate transient receptor potential cation channel, subfamily V, member 1-positive nerve terminals. The effects of resiniferatoxin on postincisional measures of pain were assessed through postoperative day 10 (n = 51). Transcriptomic changes in the dorsal spinal cord, with and without peripheral transient receptor potential cation channel, subfamily V, member 1-positive nerve terminal inactivation, were assessed by RNA sequencing (n = 22). RESULTS: Peripherally administered resiniferatoxin increased thermal withdrawal latency by at least twofold through postoperative day 4, increased mechanical withdrawal threshold by at least sevenfold through postoperative day 2, and decreased guarding score by 90% relative to vehicle control (P < 0.05). Surgical incision induced 70 genes in the dorsal horn, and these changes were specific to the ipsilateral dorsal horn. Gene induction with surgical incision persisted despite robust analgesia from resiniferatoxin pretreatment. Many of the genes induced were related to microglial activation, such as Cd11b and Iba1. CONCLUSIONS: A single subcutaneous injection of resiniferatoxin before incision attenuated both evoked and nonevoked measures of postoperative pain. Surgical incision induced transcriptomic changes in the dorsal horn that persisted despite analgesia with resiniferatoxin, suggesting that postsurgical pain signals can be blocked without preventing transcription changes in the dorsal horn.

RNA-Seq investigations of human post-mortem trigeminal ganglia.

Background The trigeminal ganglion contains neurons that relay sensations of pain, touch, pressure, and many other somatosensory modalities to the central nervous system. The ganglion is also a reservoir for latent herpes virus 1 infection. To gain a better understanding of molecular factors contributing to migraine and headache, transcriptome analyses were performed on postmortem human trigeminal ganglia. Methods RNA-Seq measurements of gene expression were conducted on small sub-regions of 16 human trigeminal ganglia. The samples were also characterized for transcripts derived from viral and microbial genomes. Herpes simplex virus 1 (HSV-1) antibodies in blood were measured using the luciferase immunoprecipitation assay. Results Observed molecular heterogeneity could be explained by sampling of anatomically distinct sub-regions of the excised ganglia consistent with neurally-enriched and non-neural, i.e. Schwann cell, enriched subregions. The levels of HSV-1 transcripts detected in trigeminal ganglia correlated with blood levels of HSV-1 antibodies. Multiple migraine susceptibility genes were strongly expressed in neurally-enriched trigeminal samples, while others were expressed in blood vessels. Conclusions These data provide a comprehensive human trigeminal transcriptome and a framework for evaluation of inhomogeneous post-mortem tissues through extensive quality control and refined downstream analyses for RNA-Seq methodologies. Expression profiling of migraine susceptibility genes identified by genetic association appears to emphasize the blood vessel component of the trigeminovascular system. Other genes displayed enriched expression in the trigeminal compared to dorsal root ganglion, and in-depth transcriptomic analysis of the KCNK18 gene underlying familial migraine shows selective neural expression within two specific populations of ganglionic neurons. These data suggest that expression profiling of migraine-associated genes can extend and amplify the underlying neurobiological insights obtained from genetic association studies.

Comparative Analysis of Dorsal Root, Nodose and Sympathetic Ganglia for the Development of New Analgesics.

Interoceptive and exteroceptive signals, and the corresponding coordinated control of internal organs and sensory functions, including pain, are received and orchestrated by multiple neurons within the peripheral, central and autonomic nervous systems. A central aim of the present report is to obtain a molecularly informed basis for analgesic drug development aimed at peripheral rather than central targets. We compare three key peripheral ganglia: nodose, sympathetic (superior cervical), and dorsal root ganglia in the rat, and focus on their molecular composition using next-gen RNA-Seq, as well as their neuroanatomy using immunocytochemistry and in situ hybridization. We obtained quantitative and anatomical assessments of transmitters, receptors, enzymes and signaling pathways mediating ganglion-specific functions. Distinct ganglionic patterns of expression were observed spanning ion channels, neurotransmitters, neuropeptides, G-protein coupled receptors (GPCRs), transporters, and biosynthetic enzymes. The relationship between ganglionic transcript levels and the corresponding protein was examined using immunohistochemistry for select, highly expressed, ganglion-specific genes. Transcriptomic analyses of spinal dorsal horn and intermediolateral cell column (IML), which form the termination of primary afferent neurons and the origin of preganglionic innervation to the SCG, respectively, disclosed pre- and post-ganglionic molecular-level circuits. These multimodal investigations provide insight into autonomic regulation, nodose transcripts related to pain and satiety, and DRG-spinal cord and IML-SCG communication. Multiple neurobiological and pharmacological contexts can be addressed, such as discriminating drug targets and predicting potential side effects, in analgesic drug development efforts directed at the peripheral nervous system.

Haploinsufficiency of the brain-derived neurotrophic factor gene is associated with reduced pain sensitivity.

Rare pain-insensitive individuals offer unique insights into how pain circuits function and have led to the development of new strategies for pain control. We investigated pain sensitivity in humans with WAGR (Wilms tumor, aniridia, genitourinary anomaly, and range of intellectual disabilities) syndrome, who have variably sized heterozygous deletion of the 11p13 region. The deletion region can be inclusive or exclusive of the brain-derived neurotrophic factor (BDNF) gene, a crucial trophic factor for nociceptive afferents. Nociceptive responses assessed by quantitative sensory testing demonstrated reduced pain sensitivity only in the WAGR subjects whose deletion boundaries included the BDNF gene. Corresponding behavioral assessments were made in heterozygous Bdnf knockout rats to examine the specific role of Bdnf. These analogous experiments revealed impairment of Aδ- and C-fiber-mediated heat nociception, determined by acute nociceptive thermal stimuli, and in aversive behaviors evoked when the rats were placed on a hot plate. Similar results were obtained for C-fiber-mediated cold responses and cold avoidance on a cold-plate device. Together, these results suggested a blunted responsiveness to aversive stimuli. Our parallel observations in humans and rats show that hemizygous deletion of the BDNF gene reduces pain sensitivity and establishes BDNF as a determinant of nociceptive sensitivity.

Pain control through selective chemo-axotomy of centrally projecting TRPV1+ sensory neurons.

Agonists of the vanilloid receptor transient vanilloid potential 1 (TRPV1) are emerging as highly efficacious nonopioid analgesics in preclinical studies. These drugs selectively lesion TRPV1+ primary sensory afferents, which are responsible for the transmission of many noxious stimulus modalities. Resiniferatoxin (RTX) is a very potent and selective TRPV1 agonist and is a promising candidate for treating many types of pain. Recent work establishing intrathecal application of RTX for the treatment of pain resulting from advanced cancer has demonstrated profound analgesia in client-owned dogs with osteosarcoma. The present study uses transcriptomics and histochemistry to examine the molecular mechanism of RTX action in rats, in clinical canine subjects, and in 1 human subject with advanced cancer treated for pain using intrathecal RTX. In all 3 species, we observe a strong analgesic action, yet this was accompanied by limited transcriptional alterations at the level of the dorsal root ganglion. Functional and neuroanatomical studies demonstrated that intrathecal RTX largely spares susceptible neuronal perikarya, which remain active peripherally but unable to transmit signals to the spinal cord. The results demonstrate that central chemo-axotomy of the TRPV1+ afferents underlies RTX analgesia and refine the neurobiology underlying effective clinical use of TRPV1 agonists for pain control.

A systems approach for discovering linoleic acid derivatives that potentially mediate pain and itch.

Chronic pain and itch are common hypersensitivity syndromes that are affected by endogenous mediators. We applied a systems-based, translational approach to predict, discover, and characterize mediators of pain and itch that are regulated by diet and inflammation. Profiling of tissue-specific precursor abundance and biosynthetic gene expression predicted that inflamed skin would be abundant in four previously unknown 11-hydroxy-epoxy- or 11-keto-epoxy-octadecenoate linoleic acid derivatives and four previously identified 9- or 13-hydroxy-epoxy- or 9- or 13-keto-epoxy-octadecenoate linoleic acid derivatives. All of these mediators were confirmed to be abundant in rat and human skin by mass spectrometry. However, only the two 11-hydroxy-epoxy-octadecenoates sensitized rat dorsal root ganglion neurons to release more calcitonin gene-related peptide (CGRP), which is involved in pain transmission, in response to low pH (which mimics an inflammatory state) or capsaicin (which activates ion channels involved in nociception). The two 11-hydroxy-epoxy-octadecenoates share a 3-hydroxy-Z-pentenyl-E-epoxide moiety, thus suggesting that this substructure could mediate nociceptor sensitization. In rats, intradermal hind paw injection of 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate elicited C-fiber-mediated sensitivity to thermal pain. In a randomized trial testing adjunctive strategies to manage refractory chronic headaches, reducing the dietary intake of linoleic acid was associated with decreases in plasma 11-hydroxy-12,13-trans-epoxy-(9Z)-octadecenoate, which correlated with clinical pain reduction. Human psoriatic skin had 30-fold higher 9-keto-12,13-trans-epoxy-(10E)-octadecenoate compared to control skin, and intradermal injection of this compound induced itch-related scratching behavior in mice. Collectively, these findings define a family of endogenous mediators with potential roles in pain and itch.