Temporary Nerve Block Research Articles

Abstract PO-050: Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis

Abstract The impact of peripheral neurons on carcinogenesis is an understudied process in head and neck cancer. Peripheral nerve innervation is increased in preclinical models of oral cancer; sympathectomy resulted in smaller, less invasive cancers. Local neurotransmitter release from peripheral neurons innervating cancer has been linked to cancer growth and immune suppression. We hypothesize that local neurotransmitter release from trigeminal sensory neurons (TGNs) and sympathetic postganglionic neurons promotes cancer proliferation and suppresses the immune response. We used real-time PCR to determine RNA expression in human oral cancer cell lines and a colorimetric cell proliferation assay to quantify cancer cell growth in response to the sympathetic and sensory neurotransmitters. Oral cancer cell lines, HSC-3 and SCC-4, overexpress the norepinephrine (NE) receptor, adrenergic receptor beta 2 (ADRβ2) and sensory neurotransmitter receptors, receptor activity modifying protein1 (RAMP1) and neurokinin1 receptor (NK1R) compared to the non-tumorigenic cell line, HaCaT. Treatment with sympathetic (NE) and sensory (CGRP, substance p) neurotransmitters for 48 hours significantly increased cell proliferation in HSC-3 (>2-fold) and SCC-4 (>1.5-fold) cell lines, but had no effect on HaCaT cell proliferation. Using optical clearing and immunohistochemistry, we observed increased PGP9.5+ neuronal fiber density in the microenvironment using high-resolution microscopy in a tongue oral cancer xenograft mouse model. In vitro we measured a 44.6 ± 1.14% increase in TGN sprouting after 48-hour exposure to conditioned media from the HSC-3, compared to conditioned media from HaCaT. Lastly, HSC-3 cell culture supernatant injection (50 µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10 ± 1.2 % of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50 µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) compared to HSC-3 supernatant without pharmacologic nerve block. These results together suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction might improve strategies to treat head ad neck cancer by targeting peripheral neurons in the cancer microenvironment. Citation Format: Megan A. Atherton, Marci L. Nilsen, Nicole N. Scheff. Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-050.

(195) Trigeminal Ganglia Neurons Support Oral Carcinogenesis through Neurotransmitter Release in a Mouse Model of Tongue Squamous Cell Carcinoma

Tongue cancer patients suffer severe, chronic pain driven by oral cancer-induced activation of sensory neurons innervating the oral cavity. However, the impact of sensory neurotransmission on oral carcinogenesis is understudied. Local neurotransmitter release from sensory neurons innervating the cancer has been linked to cancer growth and immune suppression. We hypothesize that oral cancer-induced neurotransmitter release from trigeminal sensory neurons (TGNs) promotes oral cancer proliferation and suppresses the immune response. To investigate sensory neuron subtypes that may impact cancer in the oral cavity, we characterized TGNs that innervate the tongue using a retrograde tracer, DiI, and immunohistochemistry for neuropeptides. We used a colorimetric MTS assay kit to quantify cancer cell proliferation in response to supernatant from TGN cultures. Using flow cytometry, we tested whether pharmacologic inhibition of peripheral sensory neurons impacted inflammation in response to oral cancer. We found that a majority of TGNs innervating the tongue are peptidergic and express neurotransmitters calcitonin-gene related peptide (CGRP, 64.3±13.2%) and substance P (SP, 44.1± 9.9%). Oral cancer cell line, HSC-3, exposed to TGN culture supernatant for 48 hours had a 2.00±0.04-fold increase in proliferation compared to Dulbecco's Modified Eagle Medium (DMEM) cell culture media. HSC-3 cell culture supernatant injection (50µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10±1.2% of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66±3.4%) compared to HSC-3 supernatant without pharmacologic nerve block. These results suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction may improve strategies to treat oral cancer and pain by targeting sensory neurons.

Abstract 1057: Characterization and functional consequences of neuronal invasion in oral cancer

Abstract Most oral cancer patients suffer severe, chronic pain promoted by oral cancer-induced changes in sensory nerves innervating the oral cavity. However, the impact of sensory neurons on carcinogenesis beyond nociceptive signaling is an understudied problem in oral cancer. We hypothesize that oral cancer and sensory neurons interact, such that, oral cancer induces sensitization, sprouting and plasticity in neurons, and efferent neuronal activity promotes oral carcinogenesis. Using an oral cancer subcutaneous xenograft model, we tested the impact of peripheral nerve presence on squamous cell carcinoma tumor growth in the mouse hindpaw. Mice that received a sciatic nerve axotomy one week prior to HSC-3 oral cancer cell inoculation developed significantly smaller tumors (28.23 ± 6.6 % of total paw area) compared to mice that received sham surgery (50.71 ± 2.48 %). These data suggest that peripheral neuron innervation supports oral cancer growth. Additionally, neuropeptides are released from primary afferent neurons and suppression of the immune response has been linked to peripheral neurotransmission. Using flow cytometry, we tested whether inhibition of peripheral sensory neurons impacted inflammation in response to oral cancer. Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to injection of supernatant from HSC-3 oral cancer cells resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) into the tongue compared to HSC-3 supernatant with intact nerve function (9.10 ± 1.2 %). These results suggest neuron-mediated suppression of cancer-evoked inflammation and support the premise that neurons and neuronal activity modulates the oral cancer microenvironment. High-resolution imaging of neuronal innervation into tongue cancer and its remodeling in response to pathological changes is challenging due to dense neuronal innervation in the tongue. We utilized a recently developed optical clearing protocol (CLARITY) that preserves protein integrity and three-dimensional structure of tissue and high-resolution microscopy to visualize and characterize sensory neuron innervation in thick 1 mm tongue sections from an orthotopic xenograft mouse model of oral cancer. In a preliminary study of neuronal innervation in a tongue HSC-3 xenograft model, we observed that PGP9.5+ neuronal fiber density was greater in the cancer environment and diminished with distance from the cancer. These data suggest that oral cancer induces plasticity and sprouting of innervating peripheral neurons. A better understanding of the nerve-cancer interaction has potential for development of new therapeutic strategies to treat oral cancer and oral cancer pain by targeting sensory neurons. Citation Format: Nicole N. Scheff, Richard Klares, Brian L. Schmidt. Characterization and functional consequences of neuronal invasion in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1057.

Abstract No. 467 - Temporary nerve block for pain management in thermal ablation of the liver

Abstract No. 467 - Temporary nerve block for pain management in thermal ablation of the liver

Temporary Nerve Block at Selected Digits Revealed Hand Motor Deficits in Grasping Tasks.

Peripheral sensory feedback plays a crucial role in ensuring correct motor execution throughout hand grasp control. Previous studies utilized local anesthesia to deprive somatosensory feedback in the digits or hand, observations included sensorimotor deficits at both corticospinal and peripheral levels. However, the questions of how the disturbed and intact sensory input integrate and interact with each other to assist the motor program execution, and whether the motor coordination based on motor output variability between affected and non-affected elements (e.g., digits) becomes interfered by the local sensory deficiency, have not been answered. The current study aims to investigate the effect of peripheral deafferentation through digital nerve blocks at selective digits on motor performance and motor coordination in grasp control. Our results suggested that the absence of somatosensory information induced motor deficits in hand grasp control, as evidenced by reduced maximal force production ability in both local and non-local digits, impairment of force and moment control during object lift and hold, and attenuated motor synergies in stabilizing task performance variables, namely the tangential force and moment of force. These findings implied that individual sensory input is shared across all the digits and the disturbed signal from local sensory channel(s) has a more comprehensive impact on the process of the motor output execution in the sensorimotor integration process. Additionally, a feedback control mechanism with a sensation-based component resides in the formation process for the motor covariation structure.

Gait compensations in rats after a temporary nerve palsy quantified using temporo-spatial and kinematic parameters.

The aim of this work was to test a method for measuring the gait of rats with sufficient sensitivity to detect subtle locomotor changes due to pathology, injury and recovery. The gait of female Sprague-Dawley rats was assessed using an optical motion tracking system and the DigiGait™ imaging system during normal locomotion, shortly after temporary nerve block to the left hind limb and after full recovery. The effect of low treadmill speeds (10-30 cm/s) was initially investigated. Significant changes were detected in the spatiotemporal gait parameters, consistent with those previously reported. The overall ranges of motion in the hip, knee and ankle joints were 37.5° (±7.1°), 50.2° (±9.4°) and 61.6° (±9.1°) and did not appear to change with speed, indicating that for low speed variations, kinematic comparisons across speeds may be possible. Following the induction of a temporary sciatic nerve block, the range of motion of the left ankle and knee during swing decreased by 23° and 33°, respectively (p<0.05). A compensatory change of a greater range of motion at the hip was noted in the contralateral limb (p<0.01). 90 min post injection, most of the gait parameters had returned to normal, however, minor walking deficits were still present. Discriminant analysis showed that a combination of dynamic and kinematic parameters provides a more robust method for the classification of gait changes. This more detailed method, employing both dynamic analysis and joint kinematics simultaneously, was found to be a reliable approach for the quantification of gait in rats.

Treatment of Spasmodic Dysphonia by Means of Resection of the Recurrent Laryngeal Nerve

Over the last twelve years 44 patients suffering from spasmodic dysphonia have been seen at Huddinge hospital. There were 34 women and 10 men. 22 of these patients were for various reasons not regarded as candidates for surgery. The opposite applied to the remaining 22 patients. The outcome of a temporary nerve block guided us (the surgeons and the patients) whether to operate or not. 12 patients were thus operated upon by means of resection of the right recurrent laryngeal nerve. The immediate post-operative result was excellent in 11 of 12 patients but the voice deteriorated in 4 patients. They had revision surgery with a successful result in 3 of them. Also the other 7 patients deteriorated to some extent but not to the degree that revision surgery was necessary. It is our hypothesis that recurrencies are caused by reinnervation of the sectioned nerve.

Procedures for the selection of spastic dysphonia patients for recurrent laryngeal nerve section.

Patients with spastic dysphonia were studied to identify the characteristics of patients benefiting from recurrent laryngeal nerve (RLN) surgery. Studies included laryngeal video recordings made during speech, measures of frequency and amplitude variations during extended phonation, the effects of altered auditory feedback on dysfluencies, and temporary unilateral RLN block. Two patients had vocal fold adductor spasms during connected speech, had increased acoustic phonatory tremor in frequency, were not benefited by altered auditory feedback, and responded favorably to nerve block. Subsequent RLN section improved the speech of both patients. Other patients had speech blocks, repetitions and prolongations, vocal fold tremors during phonation, reduced dysfluencies during altered auditory feedback, and were not benefited by temporary nerve block. Two subtypes of spastic dysphonic patients are proposed.