CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia

Abstract

We studied, in male Sprague Dawley rats, the role of the cognate hyaluronan receptor, CD44 signaling in the antihyperalgesia induced by high molecular weight hyaluronan (HMWH). Low molecular weight hyaluronan (LMWH) acts at both peptidergic and nonpeptidergic nociceptors to induce mechanical hyperalgesia that is prevented by intrathecal oligodeoxynucleotide antisense to CD44 mRNA, which also prevents hyperalgesia induced by a CD44 receptor agonist, A6. Ongoing LMWH and A6 hyperalgesia are reversed by HMWH. HMWH also reverses the hyperalgesia induced by diverse pronociceptive mediators, prostaglandin E, epinephrine, TNFα, and interleukin-6, and the neuropathic pain induced by the cancer chemotherapy paclitaxel. Although CD44 antisense has no effect on the hyperalgesia induced by inflammatory mediators or paclitaxel, it eliminates the antihyperalgesic effect of HMWH. HMWH also reverses the hyperalgesia induced by activation of intracellular second messengers, PKA and PKC, indicating that HMWH-induced antihyperalgesia, although dependent on CD44, is mediated by an intracellular signaling pathway rather than as a competitive receptor antagonist. Sensitization of cultured small-diameter DRG neurons by prostaglandin E is also prevented and reversed by HMWH. These results demonstrate the central role of CD44 signaling in HMWH-induced antihyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain. We demonstrate that hyaluronan (HA) with different molecular weights produces opposing nociceptive effects. While low molecular weight HA increases sensitivity to mechanical stimulation, high molecular weight HA reduces sensitization, attenuating inflammatory and neuropathic hyperalgesia. Both pronociceptive and antinociceptive effects of HA are mediated by activation of signaling pathways downstream CD44, the cognate HA receptor, in nociceptors. These results contribute to our understanding of the role of the extracellular matrix in pain, and indicate CD44 as a potential therapeutic target to alleviate inflammatory and neuropathic pain.

In Vitro Nociceptor Neuroplasticity Associated with In Vivo Opioid-Induced Hyperalgesia

“…Culture of dorsal root ganglion (DRG) neurons. Primary cultures of DRG neurons were made from adult male Sprague Dawley rats (220 -235 g), as described previously (Ferrari et al, 2016(Ferrari et al, , 2018Khomula et al, 2017;Araldi et al, 2018c). In brief, under isoflurane anesthesia, rats were decapitated, and the dorsum of the vertebral column surgically removed; L 4 and L 5 DRGs were rapidly extracted, bilaterally, chilled and desheathed in Hanks' balanced salt solution (HBSS) on ice.…”

“…Microscopy and fluorescence imaging. The bright-field imaging system consisted of an inverted microscope (Eclipse TE-200, Nikon) with epifluorescence using a xenon lamp (Lambda LS, Sutter Instruments ) for excitation (Ferrari et al, 2018). Illumination was controlled by a Lambda 10 -2 filter wheel and Lambda SC Smart Shutter controllers (Sutter Instruments); an Andor Clara Interline CCD camera (Andor Technology) was used for high-resolution digital image acquisition.…”

“…Although small-, medium-, and large-sized neurons were routinely observed in the same preparation, this study focused on cells with soma diameter Ͻ30 m (small DRG neurons), predominantly representing C-type nociceptors (Harper and Lawson, 1985;Gold et al, 1996;Petruska et al, 2000Petruska et al, , 2002Woolf and Ma, 2007). After mounting a coverslip in the recording chamber, the culture medium was replaced with Tyrode's solution containing 140 mM NaCl, 4 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM glucose, 10 mM HEPES, and adjusted to pH 7.4 with NaOH; osmolarity is 310 mOsm/kg (Ferrari et al, 2016(Ferrari et al, , 2018Khomula et al, 2017;Araldi et al, 2018c). Tyrode's solution was used as external perfusion solution in the in vitro experiments.…”

“…After washout, fluorescent images were captured during the first 15 min of each experiment using a standard GFP filter set (Chroma Technology). Cells demonstrating bright fluorescence and a halo around the neuronal plasma membrane were recognized as IB4 ϩ (strongly positive, if intensity Ͼ40% of maximum for the selected field of view, and weakly positive if intensity is in the range 20 -40% of maximum), whereas those having intensity Ͻ20% of maximum were considered IB4 Ϫ (Fang et al, 2006;Khomula et al, 2013Khomula et al, , 2017Ferrari et al, 2016Ferrari et al, , 2018.…”

“…Whole-cell patch-clamp recordings in current-clamp mode were made to assess changes in the excitability of cultured DRG neurons. Commonly used protocols or their minor modifications were used to elicit action potentials (APs) (Hendrich et al, 2013;Duzhyy et al, 2015;Saloman et al, 2016;Viatchenko-Karpinski and Gu, 2016;Ferrari et al, 2018).…”