Ryoko Tamura

Muscular mechanical hyperalgesia revealed by behavioural pain test and c‐Fos expression in the spinal dorsal horn after eccentric contraction in rats

Delayed onset muscle soreness (DOMS) is quite common, but the mechanism for this phenomenon is still not understood; even the existence of muscle tenderness (mechanical hyperalgesia) has not been demonstrated in experimental models. We developed an animal model of DOMS by inducing eccentric contraction (lengthening contraction, ECC) to the extensor digitorum longus muscle (EDL), and investigated the existence of mechanical hyperalgesia in the EDL by means of behavioural pain tests (Randall‐Selitto test and von Frey hair test, applied to/through the skin on the EDL muscle) and c‐Fos expression in the spinal dorsal horn. We found that the mechanical withdrawal threshold measured with the Randall‐Selitto apparatus decreased significantly between 1 and 3 days after ECC, while that measured by von Frey hairs did not. The group that underwent stretching of the muscle only (SHAM group) showed no change in mechanical pain threshold in either test. These results demonstrated that the pain threshold of deep tissues (possibly of the muscle) decreased after ECC. c‐Fos immunoreactivity in the dorsal horn (examined 2 days after ECC/SHAM exercise) was not changed by either ECC or compression (1568 mN) to the EDL muscle by itself, but it was significantly increased by applying compression to the EDL muscle 2 days after ECC. This increase was observed in the superficial dorsal horn of the L4 segment of the ipsilateral side, and was clearly suppressed by morphine treatment (10 mg kg−1, i.p.). These results demonstrated the existence of mechanical hyperalgesia in the muscle subjected to ECC. This model could be used for future study of the neural mechanism of muscle soreness.

Norepinephrine excitation of cutaneous nociceptors in adjuvant-induced inflamed rats does not depend on sympathetic neurons

To test the contribution of sympathetic postganglionic neurons (SPGNs) to adrenergic excitation of cutaneous nociceptor activities in adjuvant-inflamed rats (AI rats), we studied the effects of norepinephrine (NE) in a group of sympathectomized AI rats. Sympathectomy was complete in 4-6 weeks by guanethidine (30-50 mg/kg). NE excited polymodal receptor units in the saphenous nerve at an incidence (25-30%) similar to that in AI rats, while CH-38083 blocked such excitation. These results show that alpha 2-adrenoceptors in SPGNs are not involved in the norepinephrine excitation of cutaneous nociceptors in AI rats.

Calcitonin gene-related peptide- and substance P-like immunoreactive fibers in the spermatic nerve and testis of the dog.

In order to determine if calcitonin gene-related peptide (CGRP) and substance P (SP) coexist in peripheral spermatic nerve fibers, we carried out a double-staining immunofluorescence study using confocal microscopy and fluorescence microscopy. CGRP- and SP-like immunoreactivity (LI) coexisted in the spermatic nerve trunk and in the single fibers running along the surface of the testis. The great majority of the SP-containing fibers also held CGRP-LI, although some fibers contained CGRP-LI without SP-LI. These observations are consistent with previous observations on testicular dorsal root ganglion neurons. Additionally, we carried out an immunogold silver staining for CGRP and found CGRP-containing nerve bundles, single nerve fibers and their nerve terminals. Some CGRP-containing nerve terminals were located very superficially in the tunica albuginea (<5 microm from the surface).

Coexistence of calcitonin gene-related peptide- and substance P-like immunoreactivity in retrogradely labeled superior spermatic neurons in the dog.

The coexistence of calcitonin gene-related peptide (CGRP) and substance P (SP) was determined in primary afferent neurons of the superior spermatic nerve of the dog. Testicular afferent neurons were visualized by retrograde labeling with the fluorescent dye fast blue. CGRP-like immunoreactivity (LI) was found in about 80% of testicular L1 and L2 dorsal root ganglion cells, and 81% of CGRP-positive neurons also contained SP. Conversely, SP-LI was found in 66% of testicular afferents, and 96% of SP-positive neurons simultaneously contained CGRP. Both CGRP- and SP-LI were observed in the whole size range of the testicular afferent neurons. No significant difference in the diameter was detected between CGRP- and SP-positive testicular afferent neurons. In contrast, the diameter of SP-positive cells was significantly smaller than that of CGRP-positive cells in the whole population of dorsal root ganglion (DRG) neurons. Compared to skin and muscle afferents, a larger population of the testicular afferents contain these peptides. This is considered to be one of the characteristic features of visceral afferents. Coexistence of CGRP and SP in testicular afferent neurons suggest a close functional relationship between these two neuropeptides in the sensory nervous system.

Immunochemical studies on the subunits of rabbit-intestinal sucrase-isomaltase complex

Purified sucrase-isomaltase complex sucrose alpha-glucohydrolase, EC 3.2.1.48 - dextrin 6-alpha-glycanohydrolase, EC 3.2.1.10) solubilized by papain from rabbit intestine was dissociated by citraconylation into its subunits, sucrase and isomaltase, which were then isolated in a form active immunologically as well as enzymatically by affinity chromatography on Sephadex G-200 and gel-filtration on Bio-gel P-300. Antibodies against the purified complex inhibited isomaltase but not sucrase and formed precipitation lines, crossing each other, with isolated sucrase and isomaltase, showing that the two enzymes differ in antigenicity from each other. By absorbing the antibodies with isolated sucrase and isomaltase, antibodies specific for isomaltase and sucrase, respectively, were obtained. Like the original antibodies, both of the specific antibodies quantitatively agglutinated microvillous vesicles. Sucrase was inhibited by neither of the antibodies. In contrast, isomaltase was greatly inhibited by the isomaltase-specific antibodies, but not by the sucrase-specific ones.

Segmental distribution of afferent neurons innervating the canine testis.

To clarify the afferent innervation of the canine scrotal contents, retrograde labeling of neurons in the dorsal root ganglia (DRG) has been carried out using two methods: (1) horseradish peroxidase (HRP) injection into the surface of the testis and epididymis; and (2) exposure of the superior spermatic nerve to a fluorescent dye (Fast blue; FB). Injections of HRP resulted in labeling of DRG cells located predominantly from T10 to L4 (87%) and, to a lesser extent, at S1-S3 (13%). Transection of the vas deferens previous to testicular injections eliminated labeling in the S1-S3 DRG, but not at thoracolumbar levels. These findings indicated that primary afferent fibers of the testis and epididymis project mainly to the DRG at higher than L4 through the superior spermatic nerve, but an additional population of the fibers also projects the sacral level through the inferior spermatic nerve. Exposure of the superior spermatic nerve to FB resulted in a similar distribution of labeled cells as compared with testicular injections of HRP after vasectomy. Labeled cells (8.1%) were also observed in the contralateral T13-L3 DRG. In both FB and HRP groups, the major part of the labeled cells was located in L1 and L2. The sizes of HRP- and FB-labeled cells were smaller than those of unlabeled cells in the L1 and L2 DRG. The cumulative frequency distribution histogram for the diameter of HRP- and FB-labeled cells could be fitted by a normal distribution.

Examination of colocalization of calcitonin gene-related peptide- and substance P-like immunoreactivity in the knee joint of the dog.

It is generally assumed that the majority of substance P (SP)-containing afferents are also immunoreactive for calcitonin gene-related peptide (CGRP). In order to determine whether this is also the case in articular afferents where the contents of these peptides are low, we carried out a double labeling study using Fast Blue (FB) as a retrograde tracer injected into the center of the knee joint cavity of the dog together with immunohistochemistry for SP and CGRP. After 7-36 days of survival, dorsal root ganglia (DRGs, L4-S1) were removed. Labeled cells were found mainly (94%) in L5 - 6 DRGs, and SP- and CGRP-like immunoreactivity was found in about 17 and 29% of FB-labeled cells, respectively. The coexistence of SP and CGRP was observed in 10.4% of articular afferents and only 62.7% of SP-positive articular neurons contained CGRP, a much lower ratio than in other afferents of the dog such as testicular afferents. Our data suggest that these peptides are not always released together and that they do not always work together in the joint under normal conditions.

Absence of mechanical hyperalgesia after exercise (delayed onset muscle soreness) in neonatally capsaicin-treated rats.

Delayed onset muscle soreness (DOMS) appears with some delay after unaccustomed, strenuous exercise, especially after lengthening contraction (LC). It is characterized by tenderness and movement related pain, namely muscular mechanical hyperalgesia. To clarify the involvement of C-fibers in this mechanical hyperalgesia, we examined whether DOMS could be induced in rats treated neonatally with capsaicin. We confirmed that a large portion of unmyelinated afferent fibers were lost in capsaicin treated rats. In these animals, LC failed to induce muscular mechanical hyperalgesia. mRNA of nerve growth factor (NGF) in the muscle, which plays a pivotal role in maintaining mechanical hyperalgesia, was upregulated in the capsaicin treated animals similar to the vehicle treated animals. These results demonstrate that C-fiber afferents are essential in transmitting the nociceptive information from exercised muscle in DOMS.

Coexistence of substance P- and CGRP-like immunoreactivity in DRG neurons of the articular nerves

Tomonari Nagata l, Asako Ueda*, Hidehiro Suzuki’, Yoriko Kawakami3 Extracellular unit discharges elicited by noxious stimulation of the sciatic nerve were recorded in the nucleus gigantocellularis (NGC). There were two groups of unit discharges with different latencies. One group had a latency of 5 ms-30 ms, the other a late latency of 90-140 ms. The stimulus intensity elicited A6 and C fiber responses in compound action potentials recorded in the proximal portion of the sciatic nerve at the end of the experiments. C-fos positive cells in NGC induced by noxious stimulation of the sciatic nerve were detected with immunohistochemical staining specific to nociceptive cell types. Nociceptin, a recently isolated novel peptide, was injected intrathecally through a catheter inserted into the foramen magnus. The discharge frequency decreased to 60-70% of the control value after nociceptin injection in both unit goups. This inhibition lasted more than 30min. Nociceptin inhibited the noxious responses of neurons in the NGC.

Immunolocalization of the 33 kD protein in the microvilli of rabbit small-intestinal epithelial cells☆

Rabbit small-intestinal microvilli isolated by a Ca2+ precipitation method contain a 33 kD protein, which has not been observed in microvilli isolated in the presence of Ca2+-chelators. The intracellular localization of this protein in rabbit intestinal epithelial cells was studied by immunofluorescence and immunoperoxidase microscopy, and was compared with that of aminopeptidase M, a well-known microvillus membrane-bound enzyme. The results obtained show that the 33 kD protein is located in the inside of the microvillus, but not in the terminal web of the epithelial cell. The protein may also be located on the basolateral surface of the cell.