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Palomino, S. M.

Publications and source records attributed to Palomino, S. M..

4 recordsLinked to original sources

T-cell distribution in the dorsal root ganglion across species, sex, and age

T-cells infiltrate somatosensory ganglia in response to nerve damage, autoimmune disease, and infection, contributing to sensory abnormalities and pain. In naive states, T-cells are rare in the rodent dorsal root ganglion (DRG) but have been reported in human and non-human primates without known relevant exposures. It remains unclear whether there are inherent evolutionary or species differences in DRG T-cell residence. Using a comparative biology approach, we investigated the frequency and distribution of T-cells in the mammalian DRG across humans, non-human primates, pigs, and rodents, and in humans investigated the contributions of sex and age. Spatial transcriptomics and immunofluorescence independently verified the robust presence of DRG T-cells at similar levels in humans, non-human primates, and pigs, but were fewer in rats and largely absent in mice. In humans, premenopausal females were more likely to have elevated DRG endoneurial T-cells than post-menopausal females or adult males. T-cells were detected in human dorsal root ganglion at as early as two months of age but were less abundant within the perineuronal niche. Most human DRG T-cells expressed distinct markers consistent with a resident memory (Trm) phenotype. We discuss the importance of studying the functional roles of DRG-resident T-cells and raise broader considerations for modelling peripheral nervous system disease.

neuroscience↗

Nav1.8 mediates peripheral-to-central nociceptive transmission independently of central presynaptic mechanisms in human DRG-spinal cord circuits

Suzetrigine, a selective Nav1.8 blocker, despite its significant role as a non-opioid analgesic, exhibits modest efficacy. The potential enhancement of Nav1.8 blocker efficacy through engagement of Nav1.8 at central terminals remains uncertain. We established a donor-derived human organotypic nociceptive circuit to determine where and how clinically relevant analgesics suppress pain signaling. Using ELISA and immunohistochemistry, we quantified calcitonin gene-related peptide (CGRP) release from acute explants of adult human spinal cord, dorsal root ganglia (DRG), and an intact DRG-spinal cord preparation preserving primary afferent anatomy and directional signaling. In isolated spinal cords, capsaicin evoked concentration-dependent spinal CGRP release without compromising viability, and clinically used analgesics inhibited it, validating the assay. In the intact circuit, the application of capsaicin to the DRG cell bodies triggered CGRP release exclusively in the spinal cord, consistent with compartmentalized neuropeptide release at central terminals. Selective Nav1.8 inhibition with suzetrigine reduced spinal CGRP release only when applied to the DRG or nerve root, not the spinal cord, indicating that Nav1.8 regulates peripheral action potential propagation rather than presynaptic transmitter release. These findings establish the first intact human pain circuit-based assay to study analgesics and demonstrate that the analgesic efficacy of Nav1.8 inhibitors is unlikely to increase with improved CNS penetration.

neuroscience↗

A molecular map of the human spinal dorsal and ventral horn defines arrangement of neuronal types and glial sex differences

The spinal cord is the gateway for somatosensory and nociceptive information to the brain and a key locus for sensory-motor integration. Studies in mice have advanced our understanding of spinal cord circuitry, and transcriptomic studies have begun to characterize the human spinal cord; however, major gaps in knowledge persist. We conducted single-nucleus sequencing of lumbar spinal cord tissue from 11 adult organ donors and annotated spinal cord cell types with high resolution spatial transcriptomics. We identified 34 spatially and transcriptionally defined neuronal classes and detected sex-specific cell types and states across multiple glial populations. Electrophysiological recordings from dorsal horn neurons revealed firing patterns for neuronal subtypes and group I mGluR-dependent plasticity. Our work defines previously unknown aspects of human spinal cord molecular anatomy and physiology.

neuroscience↗

Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9 with functional confirmation

CRISPR-Cas9 editing is now the leading method for genome editing and is being advanced for the treatment of human disease. CRIPSR editing could have many applications for treatment of neurological diseases, including pain but traditional viral vector delivery approaches have neurotoxicity limiting their use. Overcoming these issues could open the door for genome editing treatments for diseases like intractable pain where the dorsal root ganglia (DRG) would be the desired target. To this end, we describe a simple method for viral-vector-independent transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. As proof of principle, we edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successful as demonstrated by the expression of the reporters as early as two days in vitro. CRISPR-Cas9 editing was confirmed at the genome level with insertion and deletion detection system T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for intractable pain. TeaserWe describe a non-viral transfection method for CRISPR-Cas9 gene editing in human dorsal root ganglion neurons.

neuroscience↗