Manabu Shibata

Modified formalin test: characteristic biphasic pain response

&NA; A modified formalin test in mice was investigated. The pain response curve induced by 0.5% formalin was biphasic, having 2 peaks, from 0 to 5 min (first phase) and from 15 to 20 min (second phase). A low concentration of formalin was used, allowing the effects of weak analgesics to be detected. Centrally acting drugs such as narcotics inhibited both phases equally. Peripherally acting drugs such as aspirin, oxyphenbutazone, hydrocortisone and dexamethasone only inhibited the second phase. Aminopyrine and mefenamic acid which acted on both central and peripheral sites inhibited both phases, but the second phase was inhibited by lower doses. Thus, this method enables one to easily distinguish the site of action of analgesics. Furthermore, pain response in the first phase was inhibited by capsaicin‐treated desensitization and Des‐Arg9‐(Leu8)‐bradykinin (bradykinin inhibitor). The second phase was inhibited by compound 48/80 pretreatment, indomethacin and bradykinin inhibitor. Therefore, it is suggested that substance P and bradykinin participate in the manifestation of the first phase response, and histamine, serotonin, prostaglandin and bradykinin are involved in the second phase. These results indicate that the first and second phase responses induced by formalin have distinct characteristic properties, and it is a very useful method for examining pain, nociception and its modulation by pharmacological or other means.

Regulation of substance P release mediated via prejunctional histamine H3 receptors.

The involvement of the histamine H3 receptor in the regulation of substance P release in neurogenic inflammation was studied by using rat hindpaw skin. R-(-)-alpha-Methylhistamine, a specific histamine H3 receptor agonist, significantly inhibited the increased vascular permeability induced by antidromic electrical stimulation of the sciatic nerve in a dose-dependent manner at doses of 0.5-3 mg/kg (i.v.), and thioperamide (2 mg/kg i.p.), a specific histamine H3 receptor antagonist, prevented the inhibitory effect of R-(-)-alpha-methylhistamine. The antidromic stimulation also caused a significant increase in immunoreactive substance P release in the subcutaneous (s.c.) perfusate in the rat hindpaw. R-(-)-alpha-Methylhistamine (0.25-2 mg/kg) dose dependently inhibited the increase in release of immunoreactive substance P, and thioperamide (2 mg/mg i.p.) antagonized it. Perfusion of histamine (10(-3) M) elicited a significant increase of immunoreactive substance P release in the perfusate, which was reduced by R-(-)-alpha-methylhistamine and the antagonism of thioperamide was also observed. Histamine (in the presence of histamine H1 and H2 receptor antagonists) had an inhibitory effect on the electrically evoked release of immunoreactive substance P. These results strongly support the hypothesis that histamine regulates substance P release via prejunctional histamine H3 receptors that are located on peripheral endings of sensory nerves.

Role of substance P in neurogenic inflammation in the rat incisor pulp and the lower lip

Vascular permeability was significantly increased in the incisor pulp and skin of the lower lip in the rat after antidromic electrical stimulation of the inferior alveolar nerve, and this response was significantly inhibited by a substance-P antagonist. The content of substance P in the pulp and lip was also increased after stimulation. The permeability response was reduced by aspirin and bradykinin antagonists (both B1- and B2-receptor types) in the pulp and lip, indicating that prostaglandins and bradykinin may be involved. Mepyramine and methysergide inhibited the vascular response in the lip but not the pulp; the roles of histamine and serotonin differ in the two tissues. Injection of substance P into the incisor pulp and the lip skin caused dye leakage. This response was inhibited by pretreatment with compound 48/80 in the lip but not the pulp. Lip histamine content was decreased significantly after antidromic stimulation of the inferior alveolar nerve and pretreatment with compound 48/80, but was not changed in the pulp. The results suggest that substance P in the lip, after being released from the peripheral sensory-nerve endings, may act on the vascular system via histamine release from mast cells; but in the pulp may cause vascular response directly because of the scarcity of mast cells.

ATP-sensitive K+ channels mediate regulation of substance P release via the prejunctional histamine H3 receptor

Perfusion of histamine (10(-3) M) elicited a significant increase of immunoreactive substance P release in the subcutaneous perfusate in the rat hindpaw. The active L-enantiomer of cromakalim, lemakalim (50 micrograms/kg, i.v.), a selective K+ channel activator, significantly inhibited the immunoreactive substance P release. Glibenclamide (10 mg/kg, i.v.), an ATP-sensitive K+ channel blocker, abolished the response to lemakalim on the release of immunoreactive substance P. R(-)-alpha-methylhistamine (1 mg/kg, i.v.), a specific histamine H3 receptor agonist, significantly inhibited the release of immunoreactive substance P. Glibenclamide (10 mg/kg, i.v.) antagonized the inhibitory effect of R(-)-alpha-methylhistamine. Tetraethylammonium (10 mg/kg, i.p.), a K+ channel blocker, also reduced the inhibitory effect significantly. These results suggest that the inhibition of substance P release from sensory nerve endings via prejunctional histamine H3 receptors may be achieved by activating the ATP-sensitive K+ channel coupled to the histamine H3 receptor in the rat skin.

Naloxone prevents the analgesic action of α-MSH in mice

α-MSH (0.1, 1, 10 μg) was administered intracerebroventricularly and its action on pain sensitivity was investigated by the hot-plate method in mice. α-MSH produced dose-dependent analgesia and this analgesic effect was prevented by naloxone (1 mg/kg, s.c.). It is possible that α-MSH may play a role in the mechanism of pain through endogeneous opioid systems.

The analgesia induced by intrathecal injection of ruthenium red

&NA; Intrathecally (i.t.) Administered Ruthenium red (40, 80, 160 ng) dose‐dependently inhibited formalin‐induced nociceptive response. Ruthenium red caused a significant inhibition of pain‐related behavioral responses induced by i.t. capsaicin but not by i.t. substance P. These results suggest that ruthenium red produces analgesia by inhibiting the release of neuropeptides in the spinal cord.

Effect of melatonin on pain sensitivity. II. The role of melatonin in the development of hyperalgesia.

前報において, 明暗条件の変動によって疼痛閾値が顕著に変動し, 明条件でanalgesia, 暗条件においてhyperalgesiaが出現すること, 及び暗条件におけるhyperalgesiaの主体は松果体ホルモンであるmelatoninに帰すべきことを報告した。本報告はmelatoninのhyperalgesiaの作用機序解明を目的としての種々な実験の総括である。melatoninは脳内または慢性皮下投与実験においても明確なhyperalgesiaが生ずることを確認した。しかしtetrabenazine, α-methyldopa, haloperidol, tolazoline, phentolamine, propranololなどの薬物前処置によってhyperalgesiaは消失することからdopaminergic或はadrenergic mechanismsの関与が重要であることが示唆された。melatORinはmorphine鎮痛作用を著明に増強し, 松果体摘出動物ではmorphine鎮痛は出現しなかった。またmelatoninのhyperalgesiaもnaloxoneによって拮抗されることが確認された。このことからmelatonin作用には, 体内opioidが関与しているこが推定された。

[A new method for assaying anti-inflammatory drugs. Quantitative analysis of pigment leakage into skin by Chromatoscanner CS-900 (author's transl)].

Although the pigment leakage method is one of the most conventional for determining vascular permeability, accuracy in macroscopic measurement of the diameter of the stained area with an arbitary scale leaves much to be desired. We developed a simple and beneficial method for quantitative assay using a densitometer (Chromatoscanner CS-900). Guinea pigs weighing 300 approximately 350 g were used. Formalin as a phlogistic, in a dose of 2.3 approximately 37 mg was injected intradermally in the shaved skin of the back, and 15 mg/kg of pontamine blue was then given into the femoral vein. One hour after the injection the animals were sacrificed and the skin of the back, which was stained by the leaked pigment, was stripped off and allowed to adhere to a wooden plate for 24 hours. Reflection and a zig-zag scanning technique were used to measure the volume of the leaked pigment. There was a liner relationship between the dose of formalin and the integrated values. A dose-dependent relationship was also obtained when histamine, serotonin, kallikrein and bradykinin were used as phlogistics. Representative anti-inflammatory drugs such as aspirin, hydrocortisone, oxyphenbutazone, benzydamine, diclofenac sodium, sodium salicylate and aminopyrine depressed the leakage due to formalin. Depression of leakage by aspirin in a dose of 400 mg/kg was the most remarkable. Pigment leakage elicited by histamine, serotonin, kallikrein and bradykinin was examined on the same individual animal. Aspirin more than the other agents depressed the leakages due to bradykinin and kallikrein. Hydrocortisone and oxyphenbutazone depressed the leakage due to bradykinin, serotonin and histamine, but enhanced that due to kallikrein. The results obtained were consistent with those of a previous study and as this method is simple and more reliable, it is applicable for assay of anti-inflammatory compounds.