Spinal Autofluorescent Flavoprotein Imaging in a Rat Model of Nerve Injury-Induced Pain and the Effect of Spinal Cord Stimulation

Nerve injury may cause neuropathic pain, which involves hyperexcitability of spinal dorsal horn neurons. The mechanisms of action of spinal cord stimulation (SCS), an established treatment for intractable neuropathic pain, are only partially understood. We used Autofluorescent Flavoprotein Imaging (AFI) to study changes in spinal dorsal horn metabolic activity. In the Seltzer model of nerve-injury induced pain, hypersensitivity was confirmed using the von Frey and hotplate test. 14 Days after nerve-injury, rats were anesthetized, a bipolar electrode was placed around the affected sciatic nerve and the spinal cord was exposed by a laminectomy at T13. AFI recordings were obtained in neuropathic rats and a control group of naive rats following 10 seconds of electrical stimulation of the sciatic nerve at C-fiber strength, or following non-noxious palpation. Neuropathic rats were then treated with 30 minutes of SCS or sham stimulation and AFI recordings were obtained for up to 60 minutes after cessation of SCS/sham. Although AFI responses to noxious electrical stimulation were similar in neuropathic and naive rats, only neuropathic rats demonstrated an AFI-response to palpation. Secondly, an immediate, short-lasting, but strong reduction in AFI intensity and area of excitation occurred following SCS, but not following sham stimulation. Our data confirm that AFI can be used to directly visualize changes in spinal metabolic activity following nerve injury and they imply that SCS acts through rapid modulation of nociceptive processing at the spinal level

Spinal Autofluorescent Flavoprotein Imaging in a Rat Model of Nerve Injury-Induced Pain and the Effect of Spinal Cord Stimulation

Nerve injury may cause neuropathic pain, which involves hyperexcitability of spinal dorsal horn neurons. The mechanisms of action of spinal cord stimulation (SCS), an established treatment for intractable neuropathic pain, are only partially understood. We used Autofluorescent Flavoprotein Imaging (AFI) to study changes in spinal dorsal horn metabolic activity. In the Seltzer model of nerve-injury induced pain, hypersensitivity was confirmed using the von Frey and hotplate test. 14 Days after nerve-injury, rats were anesthetized, a bipolar electrode was placed around the affected sciatic nerve and the spinal cord was exposed by a laminectomy at T13. AFI recordings were obtained in neuropathic rats and a control group of naýve rats following 10 seconds of electrical stimulation of the sciatic nerve at C-fiber strength, or following non-noxious palpation. Neuropathic rats were then treated with 30 minutes of SCS or sham stimulation and AFI recordings were obtained for up to 60 minutes after cessation of SCS/sham. Although AFI responses to noxious electrical stimulation were similar in neuropathic and naýve rats, only neuropathic rats demonstrated an AFI-response to palpation. Secondly, an immediate, short-lasting, but strong reduction in AFI intensity and area of excitation occurred following SCS, but not following sham stimulation. Our data confirm that AFI can be used to directly visualize changes in spinal metabolic activity following nerve injury and they imply that SCS acts through rapid modulation of nociceptive processing at the spinal level

The reduction of intraepidermal P2X3 nerve fiber density correlates with behavioral hyperalgesia in a rat model of nerve injury-induced pain

Skin biopsies from patients with neuropathic pain often show changes in epidermal innervation, although it remains to be elucidated to what extent such changes can be linked to a particular subgroup of nerve fibers and how these changes are correlated with pain intensity. Here, we investigated to what extent behavioral signs of hyperalgesia are correlated with immunohistochemical changes of peptidergic and non-peptidergic epidermal nerve fibers in a rat model of nerve injury-induced pain. Rats subjected to unilateral partial ligation of the sciatic nerve developed significant mechanical and thermal hyperalgesia as tested by the withdrawal responses of the ipsilateral footpad to von Frey hairs and hotplate stimulation. At day 14, epidermal nerve fiber density (IENFD) and total epidermal nerve fiber length/mm(2) (ENFL) were significantly and consistently reduced compared to the contralateral side, following testing and re-testing by two blinded observers. The expression of calcitonin gene-related peptide (CGRP), a marker for peptidergic nerve fibers, was not significantly changed on the ipsilateral side. In contrast, the expression of the P2X3 receptor, a marker for non-peptidergic nerve fibers, was not only significantly reduced but could also be correlated with behavioral hyperalgesia. When labeling both peptidergic and non-peptidergic nerve fibers with the pan-neuronal marker PGP9.5, the expression was significantly reduced, albeit without a significant correlation with behavioral hyperalgesia. In conjunction, our data suggest that the pathology of the P2X3 epidermal nerve fibers can be selectively linked to neuropathy, highlighting the possibility that it is the degeneration of these fibers that drives hyperalgesia. This article is protected by copyright. All rights reserved

Comparison of zebrafish larvae and hiPSC cardiomyocytes for predicting drug-induced cardiotoxicity in humans

Cardiovascular drug toxicity is responsible for 17% of drug withdrawals in clinical phases, half of post-marketed drug withdrawals and remains an important adverse effect of several marketed drugs. Early assessment of drug-induced cardiovascular toxicity is mandatory and typically done in cellular systems and mammals. Current in vitro screening methods allow high-throughput but are biologically reductionist. The use of mammal models, which allow a better translatability for predicting clinical outputs, is low-throughput, highly expensive, and ethically controversial. Given the analogies between the human and the zebrafish cardiovascular systems, we propose the use of zebrafish larvae during early drug discovery phases as a balanced model between biological translatability and screening throughput for addressing potential liabilities. To this end, we have developed a high-throughput screening platform that enables fully automatized in vivo image acquisition and analysis to extract a plethora of relevant cardiovascular parameters: heart rate, arrhythmia, AV blockage, ejection fraction, and blood flow, among others. We have used this platform to address the predictive power of zebrafish larvae for detecting potential cardiovascular liabilities in humans. We tested a chemical library of 92 compounds with known clinical cardiotoxicity profiles. The cross-comparison with clinical data and data acquired from human induced pluripotent stem cell cardiomyocytes calcium imaging showed that zebrafish larvae allow a more reliable prediction of cardiotoxicity than cellular systems. Interestingly, our analysis with zebrafish yields similar predictive performance as previous validation meta-studies performed with dogs, the standard regulatory preclinical model for predicting cardiotoxic liabilities prior to clinical phases

Autofluorescent flavoprotein imaging of spinal nociceptive activity

Pain arises from activation of peripheral nociceptors, and strong noxious stimuli may cause an increase in spinal excitability called central sensitization, which is likely involved in many pathological pain states. So far, it has not been achieved to simultaneously visualize in vivo both the temporal and spatial aspects of spinal activity, including central sensitization. Using autofluorescent flavoprotein imaging (AFI), an optical technique suitable for mapping activity in nervous tissue, we demonstrate a close temporal and spatial correlation of electrically evoked nociceptive input with the spinal AFI signal, representing spinal neuronal activity. The AFI signal increases linearly with stimulation intensity. Furthermore, we found that the AFI signal was much larger in intensity and size when the same electrical stimulation was applied after the induction of central sensitization by a subcutaneous capsaicin injection. Finally, innocuous palpation of the hindpaw did not evoke an AFI response in naive animals, but after capsaicin injection a strong response was obtained. This is the first report demonstrating simultaneously the temporal and spatial propagation of spinal nociceptive activity in vivo. Copyrigh

Spinal Autofluorescent Flavoprotein Imaging in a Rat Model of Nerve Injury-Induced Pain and the Effect of Spinal Cord Stimulation

Nerve injury may cause neuropathic pain, which involves hyperexcitability of spinal dorsal horn neurons. The mechanisms of action of spinal cord stimulation (SCS), an established treatment for intractable neuropathic pain, are only partially understood. We used Autofluorescent Flavoprotein Imaging (AFI) to study changes in spinal dorsal horn metabolic activity. In the Seltzer model of nerve-injury induced pain, hypersensitivity was confirmed using the von Frey and hotplate test. 14 Days after nerve-injury, rats were anesthetized, a bipolar electrode was placed around the affected sciatic nerve and the spinal cord was exposed by a laminectomy at T13. AFI recordings were obtained in neuropathic rats and a control group of naïve rats following 10 seconds of electrical stimulation of the sciatic nerve at C-fiber strength, or following non-noxious palpation. Neuropathic rats were then treated with 30 minutes of SCS or sham stimulation and AFI recordings were obtained for up to 60 minutes after cessation of SCS/sham. Although AFI responses to noxious electrical stimulation were similar in neuropathic and naïve rats, only neuropathic rats demonstrated an AFI-response to palpation. Secondly, an immediate, short-lasting, but strong reduction in AFI intensity and area of excitation occurred following SCS, but not following sham stimulation. Our data confirm that AFI can be used to directly visualize changes in spinal metabolic activity following nerve injury and they imply that SCS acts through rapid modulation of nociceptive processing at the spinal level

Mean intensity of the AFI signal (A) and area of excitation (B) following nociceptive electrical stimulation of the sciatic nerve, in naïve versus neuropathic rats (Seltzer model), on the ipsilateral (i) and contralateral (c) side of the nerve injury and nerve stimulation.

<p>Error bars indicate SEM; <i>n</i> = 20 naïve rats, <i>n</i> = 13 neuropathic rats.</p

Effect of 30 minutes SCS or sham stimulation on the intensity of the AFI signal and area of excitation in response to sciatic nerve electrical stimulation, in rats with partial ligation of the proximal sciatic nerve (Seltzer model).

<p>(A,D) Images of background fluorescence of the dorsal surface of the spinal cord at T13 of a sham (A) and SCS treated rat (D). (B,E) Area of excitation (yellow) on the ipsilateral side, directly after sham stimulation (B); after SCS, in this rat, there is no area exceeding the predefined ΔF/F level (E). (C) Time course of the intensity of the AFI signal after SCS or sham stimulation (T = 0 min), as a percentage of ΔF/F before treatment (T = -30 min), in 20×20 pixel square selections on the ipsilateral side at the L4-L6 spinal level. (F) Mean areas of excitation on the ipsilateral side directly after SCS or sham stimulation (T = 0 min), as a percentage of the areas before treatment (T = -30 min). Scale bar, 1 mm; Grayscale bar ranging from −0.75% (black) to +0.75% (white) of the 16-bit range; Error bars indicate SEM; *<i>p</i><0.05; paired <i>t</i>-tests; <i>n</i> = 7 SCS, <i>n</i> = 6 sham stimulation.</p