Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury.

BACKGROUND: Real-time quantitative PCR (qPCR) is a technique frequently used to measure changes in mRNA expression. To ensure validity of experimental findings, it is important to normalize the qPCR data to reference genes that are stable and unaffected by the experimental treatment to correct for variability among samples. Unlike in some models of neuropathic pain, reference genes for models of inflammatory injury have not been validated. This study examined four candidate reference genes in an effort to identify and validate optimal genes for normalization of transcriptional changes occurring in the dorsal horn of the spinal cord and the rostral ventromedial medulla (RVM) following intraplantar injection of complete Freund's adjuvant (CFA). RESULTS: The expression of hypoxanthine phosphoribosyltransferase 1 (Hprt1), beta-actin (Actb), mitogen-activated protein kinase 6 (Mapk6), and beta-2-microglobulin (B2m) was quantified in the dorsal horn and RVM of rats four days or two weeks after intraplantar injection of CFA or saline. The range of expression levels among these four genes differed by as much as 16-fold within the dorsal horn and the RVM. All four of these reference genes were stably expressed in both tissues and did not differ between saline and CFA-treated animals. Analyses using the statistical algorithms in geNorm and NormFinder programs determined that Mapk6 was the most stable gene and recommended the combination of Mapk6 and Actb, or Mapk6 and Hprt1, in such experimental conditions. CONCLUSIONS: This study validated the four genes Hprt1, Actb, Mapk6 or B2m and showed that any one or combination of two of them are good reference genes for normalization of mRNA expression in qPCR experiments in the spinal cord and RVM in the CFA model of inflammatory injury.

Nicotinamide Riboside Alleviates Corneal and Somatic Hypersensitivity Induced by Paclitaxel in Male Rats.

Purpose: Patients receiving chemotherapy may experience ocular discomfort and dry eye-like symptoms; the latter may be neuropathic in nature. This study assessed corneal and somatic hypersensitivity in male rats treated with paclitaxel and whether it was relieved by nicotinamide riboside (NR). Methods: Corneal sensitivity to tactile and chemical stimulation, basal tear production, and sensitivity of the hindpaw to tactile and cool stimuli were assessed before and after paclitaxel in the absence and presence of sustained treatment with 500 mg/kg per os NR. Corneal nerve density and hindpaw intraepidermal nerve fiber (IENF) density were also examined. Results: Paclitaxel-treated rats developed corneal hypersensitivity to tactile stimuli, enhanced sensitivity to capsaicin but not hyperosmolar saline, and increased basal tear production. Corneal nerve density visualized with anti-ß-tubulin or calcitonin gene-related peptide (CGRP) was unaffected. Paclitaxel induced tactile and cool hypersensitivity of the hindpaw and a loss of nonpeptidergic hindpaw IENFs visualized with anti-protein gene product (PGP) 9.5 and CGRP. NR reversed tactile hypersensitivity of the cornea without suppressing tear production or chemosensitivity; it did not alter corneal afferent density. NR also reversed tactile and cool hypersensitivity of the hindpaw without reversing the loss of hindpaw IENFs. Conclusions: These findings suggest that paclitaxel may be a good translational model for chemotherapy-induced ocular discomfort and that NR may be useful for its relief. The ability of NR to relieve somatic tactile hypersensitivity independent of changes in sensory nerve innervation suggests that reversal of terminal arbor degeneration is not critical to the actions of NR.

Nicotinamide riboside relieves paclitaxel-induced peripheral neuropathy and enhances suppression of tumor growth in tumor-bearing rats.

Nicotinamide riboside (NR) is a vitamin B3 precursor of NAD that blunts diabetic and chemotherapy-induced peripheral neuropathy in preclinical models. This study examined whether NR also blunts the loss of intraepidermal nerve fibers induced by paclitaxel, which is associated with peripheral neuropathy. The work was conducted in female rats with N-methyl-nitrosourea (MNU)-induced tumors of the mammary gland to increase its translational relevance, and to assess the interaction of NR with paclitaxel and NR's effect on tumor growth. Once daily oral administration of 200 mg/kg NR p.o. beginning with the first of 3 i.v. injections of 6.6 mg/kg paclitaxel to tumor-bearing rats significantly decreased paclitaxel-induced hypersensitivity to tactile and cool stimuli, as well as place-escape avoidance behaviors. It also blunted the loss of intraepidermal nerve fibers in tumor-bearing rats, as well as a separate cohort of tumor-naive rats. Unexpectedly, concomitant administration of NR during paclitaxel treatment further decreased tumor growth; thereafter, tumor growth resumed at the same rate as vehicle-treated controls. Administration of NR also decreased the percentage of Ki67-positive tumor cells in these rats. Once daily administration of NR did not seem to alter tumor growth or the percentage of Ki67-positive tumor cells in rats that were not treated with paclitaxel and followed for 3 months. These results further support the ability of NR to play a protective role after nerve injury. They also suggest that NR may not only alleviate peripheral neuropathy in patients receiving taxane chemotherapy, but also offer an added benefit by possibly enhancing its tumor-suppressing effects.

Nicotinamide riboside, a form of vitamin B3 and NAD+ precursor, relieves the nociceptive and aversive dimensions of paclitaxel-induced peripheral neuropathy in female rats.

Injury to sensory afferents may contribute to the peripheral neuropathies that develop after administration of chemotherapeutic agents. Manipulations that increase levels of nicotinamide adenine dinucleotide (NAD) can protect against neuronal injury. This study examined whether nicotinamide riboside (NR), a third form of vitamin B3 and precursor of NAD, diminishes tactile hypersensitivity and place escape-avoidance behaviors in a rodent model of paclitaxel-induced peripheral neuropathy. Female Sprague-Dawley rats received 3 intravenous injections of 6.6 mg/kg paclitaxel over 5 days. Daily oral administration of 200 mg/kg NR beginning 7 days before paclitaxel treatment and continuing for another 24 days prevented the development of tactile hypersensitivity and blunted place escape-avoidance behaviors. These effects were sustained after a 2-week washout period. This dose of NR increased blood levels of NAD by 50%, did not interfere with the myelosuppressive effects of paclitaxel, and did not produce adverse locomotor effects. Treatment with 200 mg/kg NR for 3 weeks after paclitaxel reversed the well-established tactile hypersensitivity in a subset of rats and blunted escape-avoidance behaviors. Pretreatment with 100 mg/kg oral acetyl-L-carnitine (ALCAR) did not prevent paclitaxel-induced tactile hypersensitivity or blunt escape-avoidance behaviors. ALCAR by itself produced tactile hypersensitivity. These findings suggest that agents that increase NAD, a critical cofactor for mitochondrial oxidative phosphorylation systems and cellular redox systems involved with fuel utilization and energy metabolism, represent a novel therapeutic approach for relief of chemotherapy-induced peripheral neuropathies. Because NR is a vitamin B3 precursor of NAD and a nutritional supplement, clinical tests of this hypothesis may be accelerated.

Increased neuronal expression of neurokinin-1 receptor and stimulus-evoked internalization of the receptor in the rostral ventromedial medulla of the rat after peripheral inflammatory injury.

This study examined possible mechanisms by which Substance P (Sub P) assumes a pronociceptive role in the rostral ventromedial medulla (RVM) under conditions of peripheral inflammatory injury, in this case produced by intraplantar (ipl) injection of complete Freund's adjuvant (CFA). In saline- and CFA-treated rats, neurokinin-1 receptor (NK1R) immunoreactivity was localized to neurons in the RVM. Four days after ipl injection of CFA, the number of NK1R-immunoreactive neurons in the RVM was increased by 30%, and there was a concomitant increase in NK1R-immunoreactive processes in CFA-treated rats. Although NK1R immunoreactivity was increased, tachykinin-1 receptor (Tacr1) mRNA was not increased in the RVM of CFA-treated rats. To assess changes in Sub P release, the number of RVM neurons that exhibited NK1R internalization was examined in saline- and CFA-treated rats following noxious heat stimulation of the hind paws. Only CFA-treated rats that experienced noxious heat stimulation exhibited a significant increase in the number of neurons showing NK1R internalization. These data suggest that tonic Sub P release is not increased as a simple consequence of peripheral inflammation, but that phasic or evoked release of Sub P in the RVM is increased in response to noxious peripheral stimulation in a persistent inflammatory state. These data support the proposal that an upregulation of the NK1R in the RVM, as well as enhanced release of Sub P following noxious stimulation, underlie the pronociceptive role of Sub P under conditions of persistent inflammatory injury.