Hiroki Fujisawa

Involvement of post-translational modification of neuronal plasticity-related proteins in hyperalgesia revealed by a proteomic analysis

To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non‐treated ones by 2‐D PAGE in the pI range of 4.0–7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI‐TOF/TOF MS and subsequent protein database searching. They included unc‐18 protein homolog 67K, collapsin response mediator protein (CRMP)‐2 and CRMP‐4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post‐translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.

Neurotropin® inhibits calpain activity upregulated by specific alternation of rhythm in temperature in the mesencephalon of rats

Aims: Neurotropin® (NTP), an analgesic for chronic pain, has antihyperalgesic effects in specific alternation of rhythm in temperature (SART)‐stressed rats. Previous studies have shown that SART stress induces hyperalgesia, as well as post‐translational modification of proteins (including substrates for calpain, a calcium‐dependent cysteine protease) in the mesencephalon of rats. To better understand the mechanism of action of NTP, we investigated whether SART stress activates calpain in the mesencephalon of rats and whether NTP inhibits this activation. Main methods: Wistar rats were exposed to SART stress for 5 days. NTP (200 NU/kg/day) was administered intraperitoneally every day from the onset of SART stress. The mechanical pain threshold was measured using the Randall‐Selitto test on the 6th day. Thereafter, the rat mesencephalon was immediately collected and calpain activity was examined using western blot analysis with a calpain cleavage site‐specific antibody. Key findings: SART stress induced hyperalgesia and increased the calpain activity in the mesencephalon of rats. In contrast, NTP treatment attenuated the hyperalgesia and prevented the increase in calpain activity in the mesencephalon of SART‐stressed rats. Interestingly, a negative correlation was identified between calpain activity and mechanical pain threshold in SART‐stressed rats treated with or without NTP. Furthermore, NTP inhibited calpain activity on mammalian uncoordinated‐18 in rat mesencephalon homogenate and Ac‐LLY‐AFC as substrates in an in vitro cell‐free system. Significance: Our data demonstrate that NTP treatment prevents SART stress‐induced calpain activation in the mesencephalon of rats and suggests that NTP‐mediated antihyperalgesia is associated with an inhibition of calpain activity in the mesencephalon.

Neurotropin attenuates local inflammatory response and inhibits demyelination induced by chronic constriction injury of the mouse sciatic nerve.

Neuropathic pain caused by nerve damage in the central and/or peripheral nervous systems is a refractory disorder and the management of such chronic pain has become a major issue. Neurotropin is a drug widely used in Japan and China to treat chronic pain. Although Neurotropin has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the mechanism of analgesic action in the peripheral nervous system remains to be elucidated. In this study, we investigated the local effects of Neurotropin on peripheral nerve damage in a chronic constriction injury (CCI) model. Neurotropin reduced mRNA expressions of IL-1β, IL-6, and TNF-α in the sciatic nerve 1 day after the injury. Activation of Erk was also inhibited locally in the Neurotropin treatment group. Since Erk activation results in demyelination along with dedifferentiation of Schwann cells, we investigated the expression level of myelin basic protein. Five days after the injury, Neurotropin attenuated the downregulation of myelin basic protein in the sciatic nerve in the CCI model. Local effects of Neurotropin around the injury site may result in discovery of new treatments for not only neuropathic pain but also demyelinating diseases and peripheral nervous system injury.