The use of ultrasonography in the reproductive evaluation of boars.

The objective was to study the use of ultrasound as a complementary test in the breeding soundness evaluation in male pigs and study the pattern of echogenicity of the testicular parenchyma in boars of different racial groups. Twenty-six adult boars from four different racial groups were used, 10 from the Piau breed (group 1), four from the commercial and finishing group (group 2), six Pietrain breed (group 3) and six from the Duroc breed (group 4). All animals were evaluated for breeding soundness evaluation and the ultrasound examination of the testicles. The groups of animals that were evaluated showed no difference in the main semen parameters that were evaluated, except for the sperm volume, concentration of the ejaculated sperm and the supravital staining; the lowest figures were for the animals from the Piau breed (group 1). In relation to the testicular biometrics, Duroc animals (group 4) had a greater scrotal width compared to the other groups. But when we assessed the intensity of pixels of the testicles, there was a difference between groups. The groups 2 (finishing animals), 3 (Pietrain) and 4 had no difference between themselves. Group 3 had greater pixel intensity in relation to group 1. Of the 26 animals studied, five showed an abnormality during ultrasound evaluation, like hydrocele, hyperechoic mass in the testicular parenchyma, cyst in the head of the epididymis and the presence of fluid in the head and tail of the epididymis. The various animal groups studied did not differ in the principal reproductive parameters evaluated, showing that despite the great variability of reproductive traits between breeds and within the same breed, the breeding soundness evaluation, the more complete it is, is essential for the selection of breeders and the ultrasonography of the reproductive system becomes an important addition in this examination.

Effect of cyclodextrin-loaded cholesterol conjugates on plasma membrane viability of Piau swine breed frozen/thawed spermatozoa.

The objective of this study was to investigate the effect of cyclodextrin-loaded cholesterol conjugates addition to freezing extenders on plasma membrane viability of frozen-thawed spermatozoa of the Piau swine breed. Twenty semen samples were used from five males. The freezing extender was based on lactose-egg yolk extender, added to 2% glycerol, 3% dimethylacetamide. The addition of cyclodextrin-loaded cholesterol conjugates was performed after centrifugation, when semen was diluted with the cooling extender. Four groups were subjected to the following treatment: without addition (group 1); 1.5 mg of cyclodextrin-loaded cholesterol/120 × 10(6) sperm (group 2); 1.5 mg of cyclodextrin-loaded cholestanol/120 × 10(6) sperm (group 3); 1.5 mg of cyclodextrin-loaded desmosterol/120 × 10(6) sperm (group 4). To check post-thawing sperm quality sperm motility and sperm morphology evaluation were used. Additionally, to check sperm viability the hypoosmotic swelling test, supravital staining, and fluorescent assay were used. The mean values recorded for total sperm motility of semen immediately after thawing were 54.5 ± 5.8, 55.5 ± 5.3, 53.7 ± 6.7, and 52.5 ± 6.6% respectively for groups one to four, without difference between themselves (p > 0.05). Regarding fluorescent assay the results were 28.3 ± 13.2, 26.9 ± 12.2, 22.2 ± 11.4, and 32.0 ± 15.3% respectively for groups one to four, also without difference between groups (p > 0,05). Similarly, complementary tests for evaluating the integrity and functionality of the plasma membrane showed no difference between treatments (p > 0.05). In conclusion, use of cyclodextrin-loaded cholesterol conjugates added to the plasma membrane of sperm did not demonstrate any additive effect on increasing and/or maintaining sperm motility.

Cryopreservation of Piau-Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes.

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post-thaw viability of Piau-breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800 g for 10 min and 2400 g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour - freezing 1 and automated cooling using a programmed freezing machine - freezing 2) were tested. Therefore, the treatments were divided into M3 - centrifugation at 2400 g for 3 min and freezing 2; M10 - centrifugation at 800 g for 10 min and freezing 2; R3 - centrifugation at 2400 g for 3 min and freezing 1; and R10 - centrifugation at 800 g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post-thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo-osmotic test (HO), sperm-egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post-thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.

Stereotactic body radiotherapy for pulmonary metastases. Prognostic factors and adverse respiratory events.

PURPOSE: The aim of this retrospective study was to evaluate the feasibility, safety, and effectiveness of stereotactic body radiotherapy (SBRT) for pulmonary metastases. PATIENTS AND METHODS: Between April 2007 and March 2011, 87 patients underwent SBRT for pulmonary metastases using the in-house Air-Bag System(TM) to obtain the four-dimensional image for treatment planning and to reduce intrafractional intrathoracic organ motion with abdominal compression to reduce the risk of radiation pneumonitis. Survival and respiratory adverse events were analyzed. RESULTS: The 2- and 3-year overall survival (OS) rates were 47 and 32 %, and the corresponding cause-specific survivals were 52 and 36 %. The 2- and 3-year OS rates were 57 and 49 % for patients in group 1, respectively, while the corresponding OS rates were 48 and 21 %, and 40 and 32 % for patients in groups 2 and 3, respectively. The 2- and 3-year local control (LC) rates were 80 and 80 %, respectively. The corresponding intrathoracic progression-free survival rates were 40 and 32 %, respectively. Concerning adverse respiratory events after SBRT for pulmonary metastases, 14 % were grade 0 (G0), 66 % G1, 13 % G2, 6 % G3, and 1 % G4. Concerning the adverse respiratory events (NCI-CTC) by grade scale, 1- and 2-year cumulative probabilities of radiation pneumonitis were 12 and 20 % for G2 and 4 and 10 % for G3/4, respectively. The mean values for cumulative V20 were 11.6 ± 8.5 %, 29.8 ± 18.6 %, and 25.7 ± 12.8 % in G0/1, G2, and G3/4, respectively. The number of pulmonary metastases that could be safely treated with SBRT was 6 PTVs (or seven gross tumor volumes) within a cumulative V20 of 30 % under the restricted intrafractional respiratory tumor motion using the Air-Bag System(TM). CONCLUSION: We propose that the number of pulmonary metastases that can be safely treated with SBRT is 6 PTVs with a cumulative V20 of 30 % under the restricted respiratory tumor motion using the Air-Bag System(TM). SBRT for pulmonary metastases offers locally effective treatment for recurrent or residual lesions after first line chemotherapy.

Intensity-modulated radiation therapy administered to a previously irradiated spine is effective and well-tolerated.

PURPOSE: This study sought to discern the clinical outcomes of intensity-modulated radiation therapy (IMRT) administered to the spine in patients who had undergone previous radiotherapy. METHODS: A total of 81 sites of 74 patients who underwent previous radiotherapy administered to the spine or peri-spine and subsequently received IMRT for the spine were analyzed in this study. The prescribed dose of 80 Gy in a biologically effective dose (BED) of α/ß = 10 (BED10) was set as the planning target volume. The constraint for the spinal cord and cauda equine was D0.1 cc ≤ 100 Gy and ≤ 150 Gy of BED for re-irradiation alone and the total irradiation dose, respectively. RESULTS: The median follow-up period was 10.1 (0.9-92.1) months after re-irradiation, while the median interval from the last day of the previous radiotherapy to the time of re-irradiation was 15.6 (0.4-210.1) months. Separately, the median prescript dose of re-irradiation was 78.0 (28.0-104.9) of BED10. The median survival time in this study was 13.9 months, with 1-, 3-, and 5-year overall survival rates of 53.7%, 29.3%, and 26.6%, respectively. The 1-, 3-, and 5-year local control rates were 90.8%, 84.0%, and 84.0%, respectively. Neurotoxicity was observed in two of 72 treatments (2.8%) assessed after re-irradiation. CONCLUSION: Re-irradiation for the spine using IMRT seems well-tolerated. Definitive re-irradiation can be a feasible treatment option in patients with the potential for a good prognosis.

Induction of the intermediate pituitary by stress: synthesis and release of a nonopioid form of beta-endorphin.

beta-Endorphin in the intermediate lobe of the pituitary gland is posttranslationally modified to produce opioid inactive peptides. Whether these are metabolites or biologically relevant products has not been known. It was found that repeated stress induces increased biosynthesis and release of beta-endorphin-like substances from the intermediate lobe of rats and that opioid-inactive N-acetylated beta-endorphin-(1-31) is selectively made and liberated. The possible role of this nonopioid product and the selective release of peptide forms are discussed.

Review article: Asia-Pacific consensus recommendations on endoscopic tissue acquisition for biliary strictures.

BACKGROUND: Pre-operative tissue diagnosis for suspected malignant biliary strictures remains challenging. AIM: To develop evidence-based consensus statements on endoscopic tissue acquisition for biliary strictures. METHODS: The initial draft of statements was prepared following a systematic literature review. A committee of 20 experts from Asia-Pacific region then reviewed, discussed, and modified the statements. Two rounds of independent voting were conducted to reach a final version. Consensus was considered to be achieved when 80% or more of voting members voted "agree completely" or "agree with some reservation." RESULTS: Eleven statements achieved consensus. The choice of tissue sampling modalities for biliary strictures depends on the clinical setting, the location of lesion, and availability of expertise. Detailed radiological and endoscopic evaluation is useful to guide the selection of appropriate tissue acquisition technique. Standard intraductal biliary brushing and/or forceps biopsy is the first option when endoscopic biliary drainage is required with an overall (range) sensitivity and specificity of 45% (26%-72%) and 99% (98%-100%), and 48% (15%-100%) and 99% (97%-100%), respectively, in diagnosing malignant biliary strictures. Probe-based confocal laser endomicroscopy and fluorescence in situ hybridisation using 4 fluorescent-labelled probes targeting chromosomes 3, 7, 17 and 9p21 locus may be added to improve the diagnostic yield. Cholangioscopy-guided biopsy and EUS-guided tissue acquisition can be considered after prior negative conventional tissue sampling with an overall (range) sensitivity and specificity of 60% (38%-88%) and 98% (83%-100%), and 80% (46%-100%) and 97% (92%-100%), respectively, in diagnosing malignant biliary strictures. CONCLUSION: These consensus statements provide evidence-based recommendations for endoscopic tissue acquisition of biliary strictures.

Feasibility of four-dimensional conformal planning for robotic radiosurgery.

Organ motion can have a severe impact on the dose delivered by radiation therapy, and different procedures have been developed to address its effects. Conventional techniques include breath hold methods and gating. A different approach is the compensation for target motion by moving the treatment beams synchronously. Practical results have been reported for robot based radiosurgery, where a linear accelerator mounted on a robotic arm delivers the dose. However, not all organs move in the same way, which results in a relative motion of the beams with respect to the body and the tissues in the proximity of the tumor. This relative motion can severely effect the dose delivered to critical structures. We propose a method to incorporate motion in the treatment planning for robotic radiosurgery to avoid potential overdosing of organs surrounding the target. The method takes into account the motion of all considered volumes, which is discretized for dose calculations. Similarly, the beam motion is taken into account and the aggregated dose coefficient over all discrete steps is used for planning. We simulated the treatment of a moving target with three different planning methods. First, we computed beam weights based on a 3D planning situation and simulated treatment with organ motion and the beams moving synchronously to the target. Second, beam weights were computed by the 4D planning method incorporating the organ and beam motion and treatment was simulated for beams moving synchronously to the target. Third, the beam weights were determined by the 4D planning method with the beams fixed during planning and simulation. For comparison we also give results for the 3D treatment plan if there was no organ motion and when the plan is delivered by fixed beams in the presence of organ motion. The results indicate that the new 4D method is preferable and can further improve the overall conformality of motion compensated robotic radiosurgery.

Pretranslational and posttranslational mechanisms for regulating beta-endorphin-adrenocorticotropin of the anterior pituitary lobe.

Stress-induced activation of secretion of ACTH and beta-endorphin (beta-END) from anterior lobe corticotrophs leads to both short term and longer term perturbation of the system. Immediately following an acute stress session, the rate of translation of the ACTH/beta-END precursor proopiomelanocortin appears accelerated by 50% and the rate of conversion of the precursor into products is doubled. These changes appear to take place at the translational and posttranslational level and reflect a better use of the preformed messenger RNA which compensates for the stress-induced peptide depletion. When the animal is subjected daily to the stress session, longer term mechanisms appear to emerge. The ACTH/beta-END stores in the gland are increased, apparently owing to an increase in transcription, as reflected by a small but significant increase in proopiomelanocortin messenger RNA. The posttranslational processing is no longer accelerated after further stress. This longer term mechanism appears to be pretranslational and to supplant the posttranslational mechanisms observed after acute stress. These two levels of control may represent different points in the regulation of this critical peptide system.

High-dose-rate interstitial brachytherapy as a monotherapy for localized prostate cancer: treatment description and preliminary results of a phase I/II clinical trial.

PURPOSE: To improve results for localized prostate cancer, a prospective clinical trial of hyperfractionated Iridium-192 high-dose-rate (HDR) brachytherapy as a monotherapy was initiated. METHODS AND MATERIALS: Between May 1995 and September 1998, 22 implants were performed on 22 patients with localized prostate cancer (T1:T2:T3:T4 = 4:6:9:3) at Osaka University Hospital. Nineteen patients, who had T3-T4 tumors or pretreatment PSA >/= 20.0 ng/mL, received hormone therapy. No patient had external beam radiation. Transperineal needle implants using real-time ultrasound guidance were performed, followed by dose optimization program. Patients were irradiated twice a day, with a time interval of more than 6 h. Total dose was 48 Gy/8 fractions/5 days or 54 Gy/9 fractions/5 days. Acute toxicity was scored using the Radiation Therapy Oncology Group (RTOG) radiation morbidity scoring criteria. Median follow-up time was 31 months. RESULTS: HDR brachytherapy as a monotherapy was well-tolerated. No significant intra- or peri-operative complications occurred. No patient experienced acute toxicity of grade 3 or more. PSA levels normalized in 95% of patients within 20 months after irradiation. Four-year clinical and biochemical relapse-free rates were 95% and 55%, respectively. CONCLUSION: Acute toxicity with this method was acceptable. Further patient accrual and longer follow-up will allow comparison to other techniques.

Radiotherapy of early tongue cancer in patients less than 40 years old.

PURPOSE: To evaluate the results of treatment for early mobile tongue cancer in patients less than 40 years. METHODS AND MATERIALS: Between January 1967 and September 1992, 70 patients less than 40 years old (young age group) with early tongue cancer (T1-2N0M0) were treated with low-dose-rate (LDR) interstitial radiotherapy at the Osaka University Hospital (OUH). All patients had a minimum 2-year follow-up (median: 13 years). External radiotherapy (median: 30 Gy) was combined in 25 cases. The treatment results were compared with those for two older age groups (middle age: 40-64 years old; old age: 65 years old or more). RESULTS: The 5-year probability of cause-specific survival (CSS) rate for the young age group was 80%, which was not significantly different from the two older groups: 81% for middle age and 71% for old age. However, male patients of young age showed significantly worse rates than those of middle and old age (p = 0.02). The 5-year local control rate for the young age group was 78%. It was not significantly different from the two older groups: 81% for middle age and 70% for old age. The incidence of regional lymph node metastasis was 32% for T1, 48% for T2, 56% for males, and 24% for females. The regional failure rate of young males was significantly higher than those of the two older groups: 32% for middle and 22% for old age (p = 0.001). CONCLUSION: The overall treatment results for patients with early tongue cancer less than 40 years old were not worse than those of older age groups. However, male gender was a risk factor for lymph node metastasis and CSS.

High dose rate versus low dose rate interstitial radiotherapy for carcinoma of the floor of mouth.

PURPOSE: Patients with cancer of the floor of mouth are treated with radiation because of functional and cosmetic reasons. We evaluate the treatment results of high dose rate (HDR) and low dose rate (LDR) interstitial radiation for cancer of the floor of mouth. METHODS AND MATERIALS: From January 1980 through March 1996, 41 patients with cancer of the floor of mouth were treated with LDR interstitial radiation using 198Au grains, and from April 1992 through March 1996 16 patients with HDR interstitial radiation. There were 26 T1 tumors, 30 T2 tumors, and 1 T3 tumor. For 21 patients treated with interstitial radiation alone, a total radiation dose of interstitial therapy was 60 Gy/10 fractions/6-7 days in HDR and 85 Gy within 1 week in LDR. For 36 patients treated with a combination therapy, a total dose of 30 to 40 Gy of external radiation and a total dose of 48 Gy/8 fractions/5-6 days in HDR or 65 Gy within 1 week in LDR were delivered. RESULTS: Two- and 5-year local control rates of patients treated with HDR interstitial radiation were 94% and 94%, and those with LDR were 75% and 69%, respectively. Local control rate of patients treated with HDR brachytherapy was slightly higher than that with 198Au grains (p = 0.113). For late complication, bone exposure or ulcer occurred in 6 of 16 (38%) patients treated with HDR and 13 of 41 (32%) patients treated with LDR. CONCLUSION: HDR fractionated interstitial brachytherapy can be an alternative to LDR brachytherapy for cancer of the floor of mouth and eliminate radiation exposure for the medical staff.

Perioperative fractionated high-dose rate brachytherapy for malignant bone and soft tissue tumors.

PURPOSE: To investigate the viability of perioperative fractionated HDR brachytherapy for malignant bone and soft tissue tumors, analyzing the influence of surgical margin. METHODS AND MATERIALS: From July 1992 through May 1996, 16 lesions of 14 patients with malignant bone and soft tissue tumors (3 liposarcomas, 3 MFHs, 2 malignant schwannomas, 2 chordomas, 1 osteosarcoma, 1 leiomyosarcoma, 1 epithelioid sarcoma, and 1 synovial sarcoma) were treated at the Osaka University Hospital. The patients' ages ranged from 14 to 72 years (median: 39 years). Treatment sites were the pelvis in 6 lesions, the upper limbs in 5, the neck in 4, and a lower limb in 1. The resection margins were classified as intracapsular in 5 lesions, marginal in 5, and wide in 6. Postoperative fractionated HDR brachytherapy was started on the 4th-13th day after surgery (median: 6th day). The total dose was 40-50 Gy/7-10 fr/4-7 day (bid) at 5 or 10 mm from the source. Follow-up periods were between 19 and 46 months (median: 30 months). RESULTS: Local control rates were 75% at 1 year and 48% in 2 years, and ultimate local control was achieved in 8 (50%) of 16 lesions. Of the 8 uncontrolled lesions, 5 (63%) had intracapsular (macroscopically positive) resection margins, and all the 8 controlled lesions (100%) had marginal (microscopically positive) or wide (negative) margins. Of the total, 3 patients died of both tumor and metastasis, 3 of metastasis alone, 1 of tumor alone, and 7 showed no evidence of disease. Peripheral nerve palsy was seen in one case after this procedure, but no infection or delayed wound healing caused by tubing or irradiation has occurred. CONCLUSION: Perioperative fractionated HDR brachytherapy is safe, well tolerated, and applicable to marginal or wide surgical margin cases.

Isolation from bovine brain of a novel analgesic pentapeptide, neo-kyotorphin, containing the Tyr-Arg (kyotorphin) unit.

A novel analgesic peptide, isolated from the bovine brain, was found to contain the Tyr-Arg (kyotorphin) unit at the C-terminal portion. This peptide, in the methanol-soluble fraction, was isolated by gel filtration and cation exchange chromatography. This peptide was termed "neo-kyotorphin" and the amino acid sequence is Thr-Ser-Lys-Tyr-Arg. The synthetically prepared neo-kyotorphin proved to have a dose-dependent analgesic effect in mice. The median analgesic dose, ED50 of neo-kyotorphin was 195 nmol/mouse (intracisternal injection), with the tail pinch test, and the potency was approximately equal that of Leu-enkephalin.

Nitric oxide synergistically potentiates interleukin-1 beta-induced increase of cyclooxygenase-2 mRNA levels, resulting in the facilitation of substance P release from primary afferent neurons: involvement of cGMP-independent mechanisms.

We previously demonstrated that cultured rat dorsal root ganglion (DRG) cells respond to stimulation with interleukin-1 beta (IL-1 beta) by releasing substance P (SP), and this response is regulated via the cyclooxygenase (COX)-2 pathway. In this study, to ascertain the interaction between nitric oxide (NO) and prostaglandins in primary afferent neurons, we investigated the effect of NO on the IL-1 beta-induced release of SP in cultured DRG cells. An NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), did not in itself evoke SP release. However, it potentiated the IL-1 beta-induced release of SP. Similarly, while SNAP did not elicit the expression of COX-2 mRNA, it potentiated the expression induced by IL-1 beta in cultured DRG cells, and this potentiation was significantly suppressed by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). Moreover, SNAP also potentiated the expression of COX-2 protein induced by IL-1 beta in cultured DRG cells. The stimulatory effect of SNAP on the IL-1 beta-induced release of SP was completely inhibited on co-incubation with a selective COX-2 inhibitor, NS-398. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a potent inhibitor of soluble guanylate cyclase, did not suppress, and a membrane-permeable cGMP analogue, 8-Br-cGMP, did not mimic the stimulatory effects of SNAP in DRG cells. These results suggest that in cultured DRG cells, NO potentiates the IL-1 beta-induced increase in COX-2 expression via a soluble guanylate cyclase-cGMP-independent pathway, resulting in facilitation of SP release. The interaction between NO and COX in primary afferent neurons might contribute to the change in nociceptive perception in inflammatory hyperalgesia.

Morphine analgesia and the bulbospinal noradrenergic system: increase in the concentration of normetanephrine in the spinal cord of the rat caused by analgesics.

1 Administration of an analgesic dose (10 mg/kg, s.c.) of morphine increased the concentration of a noradrenaline metabolite, normetanephrine (NM) in the spinal cord of normal rats. The time course of the change in the NM concentration corresponded approximately to that of the morphine analgesia. The concentration of noradrenaline was not affected.2 A similar effect on the NM concentration was also observed after the administration of pentazocine (30 mg/kg, s.c.) and nalorphine (20 mg/kg, s.c.).3 The NM increasing effect of morphine, pentazocine and nalorphine was found in the dorsal half of the spinal cord but not in the ventral half.4 The increase in the concentration of NM induced by morphine, pentazocine or nalorphine was completely suppressed by naloxone (1 mg/kg, s.c.) given 5 min before the administration of these drugs.5 When the spinal cord was transected at C1, the NM increasing effect of morphine disappeared, yet when the brain stem was transected at the inter-collicular level, the effect remained.6 In morphine-tolerant rats, the concentration of NM in the spinal cord was almost the same as that observed in normal rats, but the increase in the concentration of NM in the spinal cord after the acute administration of morphine did not take place.7 The NM concentration in the spinal cord of normal rats was not modified by aminopyrine (75 mg/kg, s.c.), chlorpromazine (10 mg/kg, s.c.), mephenesin (100 mg/kg, i.p.) or naloxone (25 mg/kg, s.c.).8 The possible relation between morphine analgesia and the descending noradrenergic neurones in the spinal cord of rats is discussed.

Involvement of endogenous nitric oxide in the mechanism of bradykinin-induced peripheral hyperalgesia.

1. When NG-nitro-L-arginine methyl ester (L-NAME, 0.1-10 nmol) or NG-monomethyl-L-arginine (L-NMMA, 10 nmol-1 mumol) was intradermally administered with bradykinin (BK, 3 nmol) into the instep of rat hind-paws, a dose-related suppression of BK-induced hyperalgesia, assessed by the paw-pressure test, was produced. 2. L-Arginine (1 mumol) but not D-arginine (1 mumol) reversed the suppressive effects of L-NAME (10 nmol) and L-NMMA (1 mumol) on BK-induced hyperalgesia. 3. Concomitant intradermal administration of BK (3 nmol) with haemoglobin (1 nmol) significantly suppressed BK-induced hyperalgesia in the paw-pressure test. The BK-induced hyperalgesia was abolished by concomitant intradermal administration of either a guanylate cyclase inhibitor, methylene blue (10 nmol), or LY83583 (1 nmol). In addition, KT5823 (1 nmol) or Rp-8-bromoguanosine-3':5'-cyclic monophosphothioate (Rp-8-Br-cGMPS; 1 nmol), an inhibitor of cyclic GMP-dependent protein kinase, also significantly suppressed BK-induced hyperalgesia. 4. The carrageenin-induced hyperalgesia was significantly attenuated by L-NAME in a dose-dependent manner. 5. L-Arginine (1 mumol), sodium nitroprusside (1 mumol), dibutyryl cyclic GMP (1 mumol) or 8-bromo cyclic GMP (1 mumol) all failed to produce any significant relieving effect on the nociceptive threshold of rodent hind-paws. Concomitant administrations of each agent with a sub-threshold dose (0.1 nmol) of BK induced significant hyperalgesia. 6. Rp-adenosine 3':5'-cyclic monophosphothioate (Rp-cAMPS; 1 nmol), an inhibitor of cyclic AMP-dependent protein kinase, significantly suppressed BK-induced mechanical hyperalgesia. Concomitant administration of forskolin (1 nmol) with 8-bromo cyclic GMP (100 nmol) induced significant hyperalgesia. 7. In the superfusion experiment of a blister base on the instep of rodent hind-paws, intradermally administered BK (3 nmol) significantly increased the outflow of both cyclic GMP and cyclic AMP from the blister base. Concomitant administrations of L-NAME (10 nmol) with BK significantly reduced the BK-induced outflow of cyclic GMP without affecting the cyclic AMP content. 8. These results suggest that the NO-cyclic GMP pathway is involved in the mechanism of BK-induced hyperalgesia, and an activation of both cyclic GMP-and cyclic AMP-second messenger system plays an important role in the production of peripherally induced mechanical hyperalgesia.