Spinal orexin-1 receptors mediate anti-hyperalgesic effects of intrathecally-administered orexins in diabetic neuropathic pain model rats

Abstract

Orexin-A and orexin-B are endogenous ligands of orexin receptors that contain orexin-1 and orexin-2. Activation of the orexinergic system can produce antinociceptive effects in acute inflammatory, mono-neuropathic, and postoperative pain animal models, though the effects of orexins on diabetic neuropathic pain have not been previously investigated. In this study, we studied the anti-hyperalgesic effects of intrathecally administered orexins in a streptozotocin-induced diabetic rat. First, dose-dependent effects were investigated by measuring hind paw withdrawal thresholds in response to noxious-heat and punctate stimuli, after which orexin levels in the cerebrospinal fluid of diabetic rats were measured and compared with those of normal rats using a radioimmunoassay method. The functional role of spinal orexin-1 receptors with the anti-hyperalgesic effects of orexins was also investigated using intrathecal pretreatment with SB-334867, a selective orexin-1 receptor antagonist. Intrathecally administered orexins produced an antinociceptive effect in diabetic rats, however, not in normal rats, though the orexin levels in the cerebrospinal fluid of diabetic rats were similar to those in normal rats. In addition, the anti-hyperalgesic effects of orexins were significantly inhibited by pretreatment with SB-334867. These findings demonstrate that the anti-hyperalgesic effects of orexins in diabetic rats are unlikely due to any direct effect by the supplement on decreased endogenous orexins in the cerebrospinal fluid and that orexin-1 receptors in the spinal cord may be involved in the modulation of nociceptive transmission in diabetic neuropathy. We conclude that the spinal orexinergic system may be a possible target for elucidating the mechanisms of diabetes-induced hyperalgesia.

Introduction

Diabetic neuropathy is one of the three most common complications of diabetes and occurs predominantly in the distal extremities as pain, which can occur either spontaneously, as a result of exposure to only mildly painful stimuli (i.e., hyperalgesia) or to stimuli not normally perceived as painful (i.e., allodynia) [3], [9]. The condition remains difficult to treat, because it is relatively resistant to standard analgesics such as opioids [2], [9].

Orexin-A and orexin-B, also known as hypocretin-1 and hypocretin-2, are composed of 33 and 28 amino acids, respectively. They are novel hypothalamic neuropeptides derived from the same 130-amino acid precursor peptide, prepro-orexin [25]. Orexins act on their targets via 2 types of G-protein-coupled receptors; orexin-1 receptors, which have an approximately 10-fold greater affinity for orexin-A than orexin-B, and orexin-2 receptors, which have a similar affinity for both [14], [25]. Their link with nociception has been extended, as studies have demonstrated that activation of the orexinergic system can produce an antinociceptive reaction in experimental animals. For example, in a rat inflammatory pain model caused by formalin injection, orexin-1 receptors were shown to mediate the analgesic action produced by intrathecal orexin-A [31]. Furthermore, our previous study [27] and one by Yamamoto et al. [33] demonstrated that intrathecal injection of orexin-A produced profound analgesic and antiallodynic effects, respectively, in the sciatic nerve of mono-neuropathic pain model rats. In addition, in a rat model of postoperative pain, both orexin-A and orexin-B were shown to induce mild but distinct antiallodynic effects when given intrathecally [8]. However, the effects of orexins on diabetic neuropathic pain have not been investigated.

Several lines of evidence suggest that the orexin-expressing neurons themselves are regulated by energy balance, while orexin expression has been shown to be stimulated by hypoglycemia in fasted rats [25]. Hypothalamic prepro-orexin mRNA levels have also been shown to be increased under both prolonged fasting [4], [25] and insulin-induced hypoglycemic [16] conditions and reduced under hyperglycemic conditions in genetically obese mice [30]. Furthermore, in studies that used fos-like immunoreactivity as a marker of neuronal activation, it has been reported that hypothalamic orexin-containing neurons are activated by insulin-induced hypoglycemia [5], [23]. Thus, orexin-expressing neurons may be a part of a group of glucose-sensitive neurons. In addition, immunohistochemical staining of orexin-containing neurons in a recent experiment demonstrated that orexins may be directly secreted into the cerebrospinal fluid [7]. Therefore, it is conceivable that orexin levels in the cerebrospinal fluid may be reduced in diabetic rats because of hyperglycemia.

In the central nervous system, orexins are localized in the narrow regions within the lateral hypothalamus and then projected to many regions involved with the spinal cord [29], while recent mapping of orexin fibers in rat brain and spinal cord tissues has revealed a differential distribution of the 2 peptides [12]. In the spinal cord dorsal horn, orexin-containing fibers are predominantly present in the superficial laminae associated with nociceptive processing [13], [17], [29]. Furthermore, detailed mapping of orexin receptor mRNA distribution by in situ hybridization has shown that the 2 orexin receptors are distributed throughout the rat brain, with different expression patterns [19], [21], [28]. In addition, both orexin-A and orexin-1 receptors have been found in dorsal root ganglion cells of the spinal cord [1]. These results strongly suggest that the orexinergic system is important for the regulation of different physiological functions, including nociception, and may have a potential role in the modulation of nociceptive transmission, especially in pain pathways.

In the present study, we investigated the effects of intrathecal orexins on both mechanical and thermal hyperalgesia induced by diabetes. Orexin levels in cerebrospinal fluid samples might be more indicative of orexin neurotransmission activity than orexin levels in brain homogenate samples, since cerebrospinal fluid orexin-A levels remain very stable and the measurement method is reliable [15]. We measured orexin levels in rat cerebrospinal fluid samples using a radioimmunoassay method to test our hypothesis that cerebrospinal fluid orexin levels are reduced in diabetic rats because of hyperglycemia. The functional roles of spinal orexin-1 receptors on the anti-hyperalgesic effects of orexins were also examined following intrathecal pretreatment with SB-334867, a selective orexin-1 receptor antagonist, to investigate the underlying mechanisms.