Animal Studies

Dry eye disease (DED) is recognized as a chronic inflammatory condition with an increase in tear osmolarity and loss of tear film integrity. DED is often accompanied by adverse ocular symptoms which are more prevalent in females than males. The basis for ocular hyperalgesia in DED remains uncertain; however, both peripheral and central neural mechanisms are implicated. A model for aqueous deficient DED, exorbital gland excision, was used to determine if activation of the purinergic receptor subtype 7, P2X7R, expressed by non-neural cells in peripheral and central trigeminal nerve pathways, contributed to persistent ocular hyperalgesia. Densitometry of trigeminal brainstem sections revealed increases in P2X7R, the myeloid cell marker Iba1, and the inflammasome, NLRP3, of estradiol-treated DED females compared to estradiol-treated sham females, while expression in DED males and DED females not given estradiol displayed minor changes. No evidence of immune cell infiltration into the trigeminal brainstem was seen in DED rats; however, markers for microglia activation (Iba1) were increased in all groups. Isolated microglia expressed increased levels of P2X7R and P2X4R, IL-1 (Ιnterleukin-1), NLRP3, and iNOS (nitric oxide synthase). Further, estradiol-treated DED females displayed greater increases in P2X7R, IL-1 and NLRP3 expression compared to untreated DED females. Orbicularis oculi muscle activity (OOemg) evoked by ocular instillation of hypertonic saline (HS) was recorded as a surrogate measure of ocular hyperalgesia and was markedly enhanced in all DED groups compared to sham rats. Systemic minocycline reduced HS-evoked OOemg in all DED groups compared to sham rats. Local microinjection in the caudal trigeminal brainstem of an antagonist for P2X7R (A804598) greatly reduced HS-evoked OOemg activity in all DE groups, while responses in sham groups were not affected. Intra-trigeminal ganglion injection of siRNA for P2X7R significantly reduced HS-evoked OOemg activity in all DED groups, while evoked responses in sham animals were not affected. These results indicated that activation of P2X7R at central and peripheral sites in trigeminal pain pathways contributed to an increase in ocular hyperalgesia and microglia activation in DED males and females. Estrogen treatment in females further amplified ocular hyperalgesia and neuroimmune responses in this model for aqueous deficient DED.

C-shapes are stereotyped movements in planarians that are elicited by diverse stimuli (e.g. acidity, excitatory neurotransmitters, psychostimulants, and pro-convulsants). Muscle contraction and seizure contribute to the expression of C-shape movements, but a causative role for pain is understudied and unclear. Here, using nicotine-induced C-shapes as the endpoint, we tested the efficacy of three classes of antinociceptive compounds – an opioid, NSAID (non-steroidal anti-inflammatory drug), and transient receptor potential ankyrin 1 (TRPA1) channel antagonist. For comparison we also tested effects of a neuromuscular blocker. Nicotine (0.1-10 mM) concentration-dependently increased C-shapes. DAMGO (1-10 µM), a selective µ-opioid agonist, inhibited nicotine (5 mM)-induced C-shapes. Naloxone (0.1-10 µM), an opioid receptor antagonist, prevented the DAMGO (1 µM)-induced reduction of nicotine (5 mM)-evoked C-shapes, suggesting an opioid receptor mechanism. C-shapes induced by nicotine (5 mM) were also reduced by meloxicam (10-100 µM), a NSAID; HC 030031 (1-10 µM), a TRPA1 antagonist; and pancuronium (10-100 µM), a neuromuscular blocker. Evidence that nicotine-induced C-shapes are reduced by antinociceptive drugs from different classes, and require opioid receptor and TRPA1 channel activation, suggest C-shape etiology involves a pain component.

Prdm12 is a conserved epigenetic transcriptional regulator that displays restricted expression in nociceptors of the developing peripheral nervous system. In mice, Prdm12 is required for the development of the entire nociceptive lineage. In humans, PRDM12 mutations cause congenital insensitivity to pain, likely because of the loss of nociceptors. Prdm12 expression is maintained in mature nociceptors suggesting a yet-to-be explored functional role in adults. Using Prdm12 inducible conditional knockout mouse models, we report that in adult nociceptors Prdm12 is no longer required for cell survival but continues to play a role in the transcriptional control of a network of genes, many of them encoding ion channels and receptors. We found that disruption of Prdm12 alters the excitability of dorsal root ganglion neurons in culture. Phenotypically, we observed that mice lacking Prdm12 exhibit normal responses to thermal and mechanical nociceptive stimuli but a reduced response to capsaicin and hypersensitivity to formalin-induced inflammatory pain. Together, our data indicate that Prdm12 regulates pain-related behavior in a complex way by modulating gene expression in adult nociceptors and controlling their excitability. The results encourage further studies to assess the potential of Prdm12 as a target for analgesic development.

The mechanism by which acetaminophen produces its analgesic effects is not fully understood. One possible mechanism is the activation of the spinal 5-hydroxytryptamine (5-HT) receptor, although direct evidence of spinal 5-HT release has not yet been reported. N-arachidonoylphenolamine (AM404), a metabolite of acetaminophen, is believed to be the key substance that contributes to the analgesic effects of acetaminophen. In this study, we examined whether acetaminophen and AM404 induce spinal 5-HT release and the mechanism through which spinal 5-HT receptor activation exerts analgesic effects in a rat formalin test in an inflammatory pain model. Spinal 5-HT release was examined by intrathecal microdialysis in conscious and freely moving rats. Acetaminophen was administered orally, and AM404 was administered intracerebroventricularly. In rat formalin tests, oral acetaminophen and intracerebroventricular AM404 induced significant spinal 5-HT release and produced analgesic effects. The analgesic effect of oral acetaminophen was partially antagonized by intrathecal administration of WAY100135 (a 5-HT receptor antagonist) and SB269970 (a 5-HT receptor antagonist). In contrast, the analgesic effect of intracerebroventricular AM404 was completely antagonized by WAY100135, while SB269970 had no effect. Our data suggest that while oral acetaminophen and intracerebroventricular AM404 activate the spinal 5-HT system, the role of the spinal 5-HT system activated by oral acetaminophen differs from that activated by intracerebroventricular AM404.

Boric acid (BA) has been used in many diseases because it increases the amount of reduced glutathione in the body and reduces oxidative damage. This study aims to investigate the effects of boric acid in cisplatin-induced neuropathy, in which oxidative stress is also effective in its pathophysiology. In this study, 8-10 weeks old, 170-190 g Wistar Albino rats were used. Each group contained seven rats (n = 35). Experimental groups consist of control, sham, neuropathy, treatment, and boric acid groups. For the neuropathy model, a single dose of cisplatin (3 mg/kg, i.p) was administered once a week for five weeks, and for the treatment group, boric acid was administered daily (100 mg/kg, intragastric) for five weeks. After drug administration, the rotarod test to evaluate motor performance, the tail-flick and hot/cold plate tests to evaluate sensory conduction states, the von Frey filament test to evaluate the mechanical allodynia, and the adhesive removal test to assess sensorimotor function were performed. The sciatic nerve's motoric conduction velocity was also assessed electrophysiologically. Oxidative stress parameters were also assessed biochemically in sciatic nerve tissue and serum. Hematoxylin and eosin staining was used to evaluate the sciatic nerve tissue histopathologically. The motor conduction velocity of the sciatic nerve, impaired by cisplatin, was increased considerably by boric acid (p < 0.05). It also reduced the latency time of the compound muscle action potential (CMAP), which was increased by cisplatin. (p < 0.05). The von Frey filament test results demonstrated increased pain sensitivity of the cisplatin group increased, and mechanical allodynia was observed. Boric acid significantly alleviated this condition (p < 0.05). In the cold plate, adhesive removal, and rotarod tests, boric acid attenuated the adverse effects of cisplatin (p < 0.05). Biochemically, BA reduced the level of MDA, which was raised by cisplatin, and significantly increased the level of SOD, which was lowered by cisplatin (p < 0.05). Histopathologically; BA reduced neuronal degeneration and vacuolization caused by cisplatin. As a consequence, it has been determined that boric acid alleviates the adverse effects of cisplatin. BA reduced the destructive effect of cisplatin by reducing oxidative stress, and this effect was verified electrophysiologically, behaviorally, and histopathologically.