Supplementary Material for: Developmental Changes in Voltage-Gated Calcium Channel α₂δ-Subunit Expression in the Canine Dorsal Root Ganglion

The voltage-gated calcium channel subunit α₂δ plays a fundamental role in propagation of excitatory signals associated with release of glutamate and neuropeptides substance P (SP) and calcitonin gene-related protein (CGRP). It can be selectively inhibited by gabapentinoids. Hence, investigation of the α₂δ subunit may predict the efficacy of gabapentinoid therapy in neuropathic pain. Since sensory processing underlies significant age-related changes, this study was conducted in order to elucidate the role of the α₂δ subunit in the sensory transmission during canine development. Dorsal root ganglia (DRG) were harvested from four spinal segments of 16 puppies and 10 adult dogs without a history of neurological signs, pain, spinal disease or orthopedic disorders. α₂δ-Subunit expression and coexpression with SP and CGRP was evaluated immunohistochemically regarding the number of immunopositive ganglion cells, staining intensity and subcellular distribution. All tested ganglia were immunopositive for α₂δ. Cell counts and expression levels were significantly lower in pups than in adult dogs (p &lt; 0.05). In the cervical segments of both groups, the number and percentage of immunopositive neurons was significantly higher than in lumbar DRG (p &lt; 0.05). Multilabeling studies in all tested animals confirmed the coexpression of α₂δ and pain peptides SP and CGRP. This anatomical study for the first time documents the involvement of α₂δ subunits in sensory signal processing in dogs. The proportion of positive neurons and the intracellular expression levels show a net increase from early postnatal life to adulthood. A significant portion of α₂δ-positive cells in the dogs exhibited C- and Aδ-phenotypes compatible with nociceptive neurons. The coexpression of α₂δ, SP and CGRP imply that these neurons are involved with peptidergic nociception. The cervicolumbar gradient of α₂δ expression in adults reflects functional differences in between forelimbs and hind limbs. These data will facilitate translational studies on neuropathic pain states in this species such as common canine nerve entrapment syndromes.电压门控钙通道亚基α₂δ（voltage-gated calcium channel subunit α₂δ）在与谷氨酸、神经肽P物质（substance P, SP）及降钙素基因相关蛋白（calcitonin gene-related protein, CGRP）释放相关的兴奋性信号传导中发挥核心作用。该亚基可被加巴喷丁类化合物（gabapentinoids）选择性抑制。因此，对α₂δ亚基的研究或可预测加巴喷丁类药物治疗神经性疼痛的疗效。由于感觉加工过程存在显著的年龄相关性变化，本研究旨在阐明α₂δ亚基在犬发育过程中感觉传导中的作用。研究采集了16只幼犬和10只成年犬的4个脊髓节段的背根神经节（dorsal root ganglia, DRG），所有受试动物均无神经症状、疼痛、脊髓疾病或骨科疾病病史。本研究采用免疫组织化学法，从免疫阳性神经节细胞数量、染色强度及亚细胞分布三个维度，评估α₂δ亚基的表达情况及其与SP、CGRP的共表达情况。所有受试背根神经节均呈α₂δ免疫阳性。幼犬的细胞计数与表达水平均显著低于成年犬（p < 0.05）。两组动物的颈段脊髓节段中，免疫阳性神经元的数量及占比均显著高于腰段背根神经节（p < 0.05）。对所有受试动物的多重标记研究证实，α₂δ与疼痛相关肽SP、CGRP存在共表达。本解剖学研究首次证实了α₂δ亚基参与犬的感觉信号加工过程。阳性神经元占比与细胞内表达水平从出生早期到成年阶段呈持续升高趋势。犬体内大量α₂δ阳性细胞呈现与伤害性神经元相符的C纤维及Aδ纤维表型。α₂δ与SP、CGRP的共表达表明，此类神经元参与肽能伤害性感受过程。成年犬α₂δ表达的颈腰梯度反映了前肢与后肢之间的功能差异。本研究数据将为该物种神经性疼痛状态（如犬常见神经卡压综合征）的转化研究提供支持。