Learning curves for robotic-assisted spine surgery: an analysis of the time taken for screw insertion, robot setting, registration, and fluoroscopy.

PURPOSE: The purpose of this study was to clarify the learning curve for robotic-assisted spine surgery. We analyzed the workflow in robotic-assisted spine surgery and investigated how much experience is required to become proficient in robotic-assisted spine surgery. METHODS: The data were obtained from consecutive 125 patients who underwent robotic-assisted screw placement soon after introducing a spine robotic system at a single center from April 2021 to January 2023. The 125 cases were divided into phases 1-5 of sequential groups of 25 cases each and compared for screw insertion time, robot setting time, registration time, and fluoroscopy time. RESULTS: There were no significant differences in age, body mass index, intraoperative blood loss, number of fused segments, operation time, or operation time per segment between the 5 phases. There were significant differences in screw insertion time, robot setting time, registration time, and fluoroscopy time between the 5 phases. The screw insertion time, robot setting time, registration time, and fluoroscopy time in phase 1 were significantly longer than those in phases 2, 3, 4, and 5. CONCLUSION: In an analysis of 125 cases after the introduction of the spine robotic system, the screw insertion time, robot setting time, registration time, and fluoroscopy time were significantly longer in the 25 cases in the period initially after introduction. The times were not significantly different in the subsequent 100 cases. Surgeons can be proficient in robotic-assisted spine surgery after their experience with 25 cases.

Accuracy of computer-assisted pedicle screw placement for adolescent idiopathic scoliosis: a comparison between robotics and navigation.

PURPOSE: To compare the accuracy of pedicle screw placement in adolescent idiopathic scoliosis (AIS) between robotics and navigation and clarify the factors that cause screw deviation when robotics is used. METHODS: Fifty consecutive patients who underwent posterior spinal fusions with computer-assisted pedicle screw placement including robotics and navigation for AIS were included. A total of 741 pedicle screws (250: Robot group, 491: Navi group) were evaluated on postoperative CT images. A rate of penetration of ≥ 2 mm was calculated as the deviation rate. After propensity score matching, we examined vertebral levels, the distance from the reference frame (RF), and the pedicle channel grade as factors for deviation. RESULTS: The deviation rate was significantly lower in the Robot group than in the Navi group (Robot group: 1.6%, Navi group: 7.5%). After propensity score matching, 22 cases were extracted. At T5-T8, the deviation rate of the Robot group was significantly lower than that of the Navi group. In the Robot group, the T2-T4 deviation rate was significantly higher than at the other vertebral levels. The distance from the RF didn't affect the deviation rate. The deviation rate of pedicle channel Grade 4 (inner diameter of less than 1 mm) was significantly higher than for the other grades. CONCLUSION: The deviation rate of robotics was 1.6%, lower than that of navigation. The narrow pedicles with an inner diameter of less than 1 mm (deviation rate: 22.2%) and the upper thoracic level (deviation rate: 14.3%) were factors related to screw deviation even when using robotics.

Safety of robotic-assisted screw placement for spine surgery: Experience from the initial 125 cases.

BACKGROUND: The present study aimed to evaluate the safety of robot-assisted screw placement in 125 cases after introducing a spinal robotics system and to identify the situations where deviation was likely to occur. METHODS: The subjects were 125 consecutive patients who underwent robotic-assisted screw placement using a spinal robotics system (Mazor X Stealth Edition, Medtronic) from April 2021 to January 2023. The 1048 screws placed with robotic assistance were evaluated. We investigated intraoperative adverse events of the robotics system and complications occurring within 30 days after surgery. We evaluated screw accuracy and deviation and compared them for vertebral levels, screw insertion methods (open traditional pedicle screw [Open-PS], cortical bone trajectory screw [CBT], percutaneous pedicle screw [PPS], and S2 alar iliac screw [S2AIS]), diagnosis, and phases of surgical cases. RESULTS: The deviation rate of robotic-assisted screw placement for spine surgery was 2.2%. Complications were reoperation due to implant-related neurological deficit in 0.8% and surgical site infection in 0.8%. There was significant difference in the deviation rate between vertebral levels. The deviation rate of the T1-T4 level was high at 10.0%. There was significant difference in the deviation rate between Open-PS, CBT, PPS, and S2AIS. The PPSs had a high deviation rate of 10.3%. The deviation rates were not significantly different between patients with and without deformity. The deviation rate did not change depending on the experience of surgical cases, and the deviation rate was favorable from the onset. CONCLUSION: Although the robotic-assisted screw placement was safe, we should be extra vigilant when placing screws in the upper thoracic region (deviation rate 10.0%) and when using PPSs (deviation rate 10.3%).

Robotics is useful for less-experienced surgeons in spinal deformity surgery.

PURPOSE: To verify whether robotics was useful for surgeons who had less experience with spinal deformity surgery. METHODS: A retrospective review was conducted of 70 consecutive patients who underwent robotic-assisted pedicle screw placements with open procedures using a spine robotic system (Mazor X Stealth Edition) at a single institution from April 2021 to April 2022. Gertzbein-Robbins grades were used to assess the deviation of the 599 pedicle screws in the postoperative CT images. The rate of Grade A was considered the perfect accuracy rate, and the rate of Grades C, D, and E was calculated as the deviation rate. The perfect accuracy rate and deviation rate were compared between the spinal deformity and the non-deformity groups. The perfect accuracy rate, deviation rate, and screw insertion time were compared in the spinal deformity cases between the expert surgeon group and the less-experienced surgeon group. RESULTS: The deviation rate of the spinal deformity group was higher than that of the non-deformity group even though there was no statistically significant difference (spinal deformity group: 2.3%, non-deformity group: 1.2%, p = 0.350). In the spinal deformity cases, there was no significant difference in the perfect accuracy rate between the expert surgeon group and the less-experienced surgeon group, but the deviation rate was significantly lower in the less-experienced surgeon group (expert surgeon group: 5.0%, less-experienced surgeon group: 0%, p = 0.008). The screw insertion time was significantly shorter in the less-experienced surgeon group. CONCLUSION: Robotics is particularly useful for surgeons with less experience in spinal deformity surgery.

Increase in pancreatic enzymes following spinal alignment changes in the thoracolumbar junction: Potential for acute pancreatitis after kyphosis correction.

BACKGROUND: Despite the identification of various risk factors for pancreatitis and hyperamylasemia following spinal surgery, no report has investigated the relationship between spinal alignment changes and elevated serum amylase levels. The purpose of this study was to investigate the relationship between spinal alignment changes and hyperamylasemia after spinal fusion. METHODS: A total of 222 patients whose serum pancreatic amylase levels were measured before and after spinal surgery from December 2017 to May 2019 were included. Inclusion criteria were (1) spinal fusion including the thoracolumbar junction (T10-L2) and (2) serum pancreatic amylase measurements before, immediately after surgery (day 0), the day after surgery (day 1), and 1 week after surgery. Ultimately, 37 patients who met the criteria were analyzed. Patients with hyperamylasemia at day 0 and/or day 1 (H group) were then compared with those without hyperamylasemia (N group). RESULTS: No significant differences in age, sex, surgical procedure, number of fused segments, intraoperative blood loss, operative time or American Society of Anesthesiologists physical status classification were observed between both groups. The H group had significantly larger preoperative thoracolumbar kyphosis (TLK) (H group: 22.6°, N group: 6.4°), postoperative TLK (H group: 16.8°, N group: 7.6°), and preoperative T12-L1 kyphosis angles (H group: 16.2°, N group: 7.9°) compared with the N group. Moreover, the H group demonstrated a significant decrease in TLK after surgery (H group: -5.8°, N group: 1.6°). CONCLUSIONS: Risk factors for hyperamylasemia included a large preoperative TLK angle and a greater postoperative decrease in TLK. Thus, decreased TLK after spinal fusion surgery should prompt careful attention to abdominal symptoms and elevated pancreatic amylase levels.

Utility of a Navigated High-Speed Drill in Robotic-Assisted Screw Placement for Spine Surgery.

Purpose To elucidate the utility of a navigated high-speed drill used after the version upgrade in surgeries assisted by a spinal robotics system. Methods The subjects were 166 patients who underwent screw placement using a spinal robotics system between April 2021 to July 2023. A significant change during the study was the introduction of a navigated high-speed drill in 80 post-upgrade cases, aimed at improving drilling accuracy. Screw accuracy was analyzed using the Gertzbein and Robbins classification on postoperative CT scans. Screws placed before (pre-upgrade group: 718 screws in 86 cases) and after the system upgrade (post-upgrade group: 747 screws in 80 cases) were compared in terms of perfect accuracy and deviation rates. Results There were no significant differences in demographics or surgical details between the two groups. No significant differences were observed in the overall perfect accuracy rate and deviation rate (2.4% pre-upgrade vs. 2.0% post-upgrade) between the two groups. For the percutaneous pedicle screw (PPS), the perfect accuracy rate was significantly higher, and the deviation rate was significantly lower in the post-upgrade group (26.1% pre-upgrade vs. 4.4% post-upgrade). Notably, the post-upgrade group achieved 100% perfect accuracy and 0% deviation for the cortical bone trajectory screw (CBT) technique. Conclusions The introduction of the navigated high-speed drill did not significantly alter the overall perfect accuracy or deviation rates for robotic-assisted screw placement. However, its use did demonstrate improved outcomes in specific techniques such as PPS and CBT, indicating its potential value in addressing skiving in robotic-assisted minimally invasive surgeries.

Nutritional Status Is Associated With Survival Following Spinal Surgery in Patients With Metastatic Spinal Tumors.

Background Preoperative and postoperative nutritional statuses are reported to influence the outcomes and complications of multidisciplinary treatment, including patient survival. However, a causal relationship between nutritional status and survival following spinal surgery has not been demonstrated in patients with metastatic spinal tumors. The present study was, therefore, designed to evaluate the correlation between the nutritional status and survival following spinal surgery in patients with metastatic spinal tumors. Methods Nutritional status was evaluated using the Japanese version of the modified Glasgow prognostic score (JmGPS), C-reactive protein-to-albumin ratio (CAR), prognostic nutrition index (PNI), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR), which were calculated from the results of preoperative laboratory tests. The survival period was defined as the interval between the day preoperative data were obtained and the day of death. Results Data from 57 of 113 consecutive surgeries were retrieved. The CAR, JmGPS, and PNI were significantly correlated with the survival period (CAR, r = -0.576, P < 0.01; JmGPS, r = -0.537, P < 0.01; PNI, r = 0.316, P = 0.02). Furthermore, patients with 0 points on the JmGPS had significantly longer survival. Using receiver operating characteristic curves, CAR cutoffs of ≥0.880 and ≤0.220 were found to be optimal in predicting the 90- and 180-day postoperative survival, respectively. Conclusions The findings of the present study indicate that preoperative assessment of the JmGPS, CAR, and PNI has utility in estimating nutritional status and predicting survival following spinal surgery in patients with metastatic spinal tumors.

Comparison of Radiographic and Patient-Reported Outcomes After Surgery in Adolescent Idiopathic Scoliosis Between Robotics and Navigation: An Analysis Using Propensity Score Matching.

Purpose This study aimed to compare the radiographic and patient-reported outcomes after surgery in adolescent idiopathic scoliosis (AIS) between robotics and navigation using propensity score matching. Methods This retrospective study involved 50 patients undergoing posterior spinal fusion for AIS between October 2016 and August 2022, utilizing navigation or robotic systems, analyzing them using propensity score matching. The evaluations included assessments using X-ray, Scoliosis Research Society 22-Item (SRS-22) Questionnaire, and CT, considering variables such as age, gender, BMI, and Lenke type. Results Post matching, 13 cases each from robotics and navigation groups were compared. No significant differences were found in the demographic variables, preoperative X-ray parameters, and preoperative SRS-22 scores between the two groups. The robotics group demonstrated a higher perfect accuracy rate (94.0% vs. 84.7%, p=0.005) and a lower deviation rate in pedicle screw placements (1.6% vs. 4.1%, p=0.223). At one year postoperatively, there were no significant differences in the X-ray parameters between both groups. Likewise, no significant differences were found in each domain of SRS-22, but function, self-image, mental health, and satisfaction scores were numerically higher in the robotics group. Conclusion The application of a spinal robotic system in AIS surgery presented enhanced screw accuracy and lower deviation rates, compared to navigation, with no significant differences observed in the X-ray parameters and each domain of SRS-22 at one year postoperatively. This suggests that, to improve patient quality of life (QOL), it is essential for robotic-assisted spine surgery to focus not only on screw accuracy but also on the development of novel robotic-assisted techniques.

Health-Related Quality of Life of Patients With Adolescent Idiopathic Scoliosis at Least 40 Years After Surgery.

STUDY DESIGN: Retrospective study. OBJECTIVE: The aim was to clarify the health-related quality of life (QOL) of patients who had adolescent idiopathic scoliosis (AIS) at a minimum of 40 years after surgery. SUMMARY OF BACKGROUND DATA: The postoperative health-related QOL of middle-aged patients with AIS has been reported to be good, but that of middle-aged and older patients with AIS has yet to be completely explored. MATERIALS AND METHODS: We included 179 patients with AIS who underwent spinal fusion(s) between 1968 and 1982. We conducted three surveys in 2009, 2014, and 2022. Patients self-administered both the Scoliosis Research Society-22 and the Roland-Morris Disability questionnaires three times (in 2009, 2014, and 2022) and the Oswestry Disability Index (ODI) questionnaire was self-administered in 2014 and 2022. We considered patients who responded to all three surveys in 2009, 2014, and 2022. RESULTS: For the Scoliosis Research Society-22 questionnaires, no significant differences were detected among the three time points (2009, 2014, and 2022) for total scores, function domain, pain domain, self-image domain, mental domain, or satisfaction domain. The results of the Roland-Morris Disability Questionnaire were also not significantly different among the surveys over time. The ODI questionnaires revealed a significant worsening of the 2022 results compared with the 2014 results. Eight patients (29.6%) who had an ODI deterioration of 10% or more had numerically fewer mobile lumbar disks than those with <10% deterioration of the ODI, although the difference did not reach statistical significance. CONCLUSION: Among patients with AIS who underwent spinal fusion(s) between 1968 and 1982, we found health-related QOL to be maintained over the last 13 years. Relatively good QOL was appreciated in this population of patients who are now middle-aged and older.

Screw Insertion Time, Fluoroscopy Time, and Operation Time for Robotic-Assisted Lumbar Pedicle Screw Placement Compared With Freehand Technique.

Introduction The purpose of this study was to clarify the superiority of robotic-assisted lumbar pedicle screw placement in terms of screw insertion time, fluoroscopy time, and operation time. Methods The subjects were 46 patients who underwent a posterior lumbar interbody fusion with an open procedure for lumbar degenerative disease from April 2021 to February 2022. The robot group contained 29 cases of screw insertion using a spine robotic system (Mazor X Stealth Edition, Medtronic Inc., Dublin, Ireland). The freehand group contained 17 cases of screw insertion with the freehand technique utilizing the conventional C-arm image guidance. The screw insertion time, fluoroscopy time, and operation time were compared between the robot and the freehand group. Results The screw insertion time did not differ significantly between the two groups (robot group: 179.0 ± 65.2 sec; freehand group: 164.2 ± 83.4 sec; p = 0.507). The fluoroscopy time was significantly shorter in the robot group (robot group: 28.3 ± 25.8 sec; freehand group: 67.5 ± 72.8 sec; p = 0.011). The fluoroscopy time per segment was also significantly shorter in the robot group (robot group: 17.8 ± 23.0 sec; freehand group: 60.2 ± 74.8 sec; p = 0.007). The operation time was significantly longer in the robot group (robot group: 249.6 ± 72.5 min; freehand group: 195.8 ± 60.1 sec; p = 0.013), but the operation time per segment did not differ significantly between the two groups (robot group: 144.1 ± 39.0 min; freehand group: 159.7 ± 34.4 min; p = 0.477). Conclusions The screw insertion time and operation time per segment were similar when employing the spine robotic system compared to the freehand technique; however, the fluoroscopy time was shorter. The fluoroscopy time per segment in the robot group was 29.6% of the time of the freehand group using the C-arm. The surgeon's radiation exposure is thought to be decreased since the spine robotic system shortens the fluoroscopy time.

Accuracy and Screw Insertion Time of Robotic-Assisted Cortical Bone Trajectory Screw Placement for Posterior Lumbar Interbody Fusion: A Comparison of Early, Middle, and Late Phases.

Introduction The purpose of this study was to evaluate robotic-assisted cortical bone trajectory (CBT) screw placement. Early, middle, and late phases of robotic-assisted CBT screw placement were compared for accuracy and screw insertion time by comparing time and accuracy in every phase.  Methods A retrospective review was conducted on the initial 40 patients who underwent spinal fusion using CBT screws in one institution from September 2021 to September 2022 utilizing a spine surgery robot system (Mazor X Stealth Edition, Medtronic Inc., Dublin, Ireland). The inclusion criterion was one- or two-level posterior lumbar interbody fusion (PLIF). Exclusion criteria were 1) patients who underwent posterior-lateral fusion in other segments, 2) patients who underwent additional decompression in other segments, 3) patients who underwent reoperation, and 4) patients with spondylolysis. The deviation of the CBT screw was evaluated on computed tomography (CT) one week after surgery using the Gertzbein-Robbins grade system. The rate of Grade A was considered the perfect accuracy rate, and the rate of penetration of 2 mm or more (Grades C, D, and E) was calculated as the deviation rate. To assess the learning curve, patients were divided into three groups. The first 10 cases were in the early phase group, the subsequent 10 cases were in the middle phase group, and the last 10 cases were in the late phase group. We compared the perfect accuracy rate, deviation rate, operative time, operative time per segment, intraoperative blood loss, registration time, and screw insertion time among the three groups. Results Thirty patients met the criteria. Overall, the perfect accuracy (Grade A) rate of the screw was 95.3% and the deviation rate was 1.4%. The perfect accuracy rate was 90.4% in the early phase, 95.5% in the middle phase, and 100% in the late phase. The deviation rate was 3.8% in the early phase, 0% in the middle phase, and 0% in the late phase, and there was no statistically significant difference between the three groups. Among the three groups, the operative time, the operative time per segment, the intraoperative blood loss, and the registration time were not significantly different. There was no significant difference in the screw insertion time among the three groups, but it decreased with experience (early phase: 156.9 ± 54.7 sec, middle phase: 139.9 ± 41.6 sec, and late phase: 106.4 ± 39.9 sec, p=0.060). The screw insertion time of the late phase tended to be shorter than that of the early phase (p=0.052). Conclusions The deviation rate of robotic-assisted CBT screw placement with one- or two-level PLIF was 1.4%, which was highly accurate. The deviation rate was 3.8% in the early phase, 0% in the middle phase, and 0% in the late phase. Although the deviation rate was low even in the early period, the screw insertion time in the early 10 cases tended to be longer than that in the late 10 cases. After passing the experience of 10 cases, this study concluded that robotic-assisted CBT screw placement was proficient.

Detection and isolation of nanobacteria-like particles from urinary stones: long-withheld data.

OBJECTIVES: To report our experimental results on detection and isolation of nanobacteria-like particles (NLP) from urinary stone samples. METHODS: From March 2001 to August 2003, 47 urinary stone samples from Japanese patients and 18 from Paraguayan patients were collected and used for compositional analysis, direct survey of NLP by scanning electron microscopy (SEM) and their cultural isolation. For the isolation, culturing was carried out using strict aseptic techniques. Dulbecco's modified Eagle medium with 10% gamma-irradiated fetal bovine serum was used based on the original method described by Kajander and Ciftçioglu. RESULTS: Positive NLP detection rates for Japanese and Paraguayan patient samples were 61.7% (29/47) and 66.7% (12/18), respectively. Positive NLP isolation rates for Japanese patient samples were 20.6% (7/34) and 20.0% (2/10) for Paraguayan patient samples. In the initial isolation, markedly different periods of incubation time were needed for each of the nine cases (6-220 days; median 36 days). Positive detection and isolation were obtained in stone samples with or without calcium phosphate. Growth modes and morphogenesis of NLP were divided into two phases; rod-shaped NLP was detected mainly as a floating form growing in culture medium and spherical NLP with a characteristic apatite shell was detected as an attached form growing on the surface of culture dishes. CONCLUSIONS: Lifeless calcifying nanoparticles can be isolated from various human urinary stones, cultured in cell culture mediums and show two characteristic growth phases.

Optimization of the Revised Tokuhashi Scoring System: New Prognostic Criteria for Metastatic Spinal Tumor in Surgical Cases.

INTRODUCTION: The revised Tokuhashi scoring system has been used to predict survival in patients with metastatic spinal tumors. Because of the rapid progress of cancer therapy, the original criteria of the revised Tokuhashi scoring system became in recent years unsuitable. The study aim was to evaluate the validity of the revised Tokuhashi scoring system in patients who underwent spinal surgery and to establish new prognostic criteria. METHODS: The study enrolled 85 patients with metastatic spinal tumors who underwent spinal surgery. The patients' survival outcomes in October 2019 were as follows: 57 patients died; 10 were alive; and 18 had unknown prognoses. The study evaluated the validity of the Tokuhashi scoring system, and established and validated the new prognostic criteria. RESULTS: The accuracies of the Tokuhashi scoring system were 66.7% in the short-term group, 60% in the midterm group, and 100% in the long-term group. Among the patients who died, the survival period and total score were significantly correlated. Total score cutoff point was six points in the patients whose predicted survival was <6 months. Total score cutoff point was eight points in the patients whose predicted survival was ≥1 year. CONCLUSIONS: As the prognosis of patients has improved in recent years, the original criteria of the revised Tokuhashi scoring system have been questioned as to their suitability to current treatments. Especially, the survival period among the patients with total scores of 7 and 8 points was not accurate. According to this study, the new prognostic criteria of the revised Tokuhashi scoring system were set to 0 to 6 points for the short-term group, 7 points for the midterm group, and 8 to 15 points for the long-term group.

Essential role of Cav3.2 T-type calcium channels in butyrate-induced colonic pain and nociceptor hypersensitivity in mice.

Given the role of Cav3.2 isoform among T-type Ca2+ channels (T-channels) in somatic and visceral nociceptive processing, we analyzed the contribution of Cav3.2 to butyrate-induced colonic pain and nociceptor hypersensitivity in mice, to evaluate whether Cav3.2 could serve as a target for treatment of visceral pain in irritable bowel syndrome (IBS) patients. Mice of ddY strain, and wild-type and Cav3.2-knockout mice of a C57BL/6J background received intracolonic administration of butyrate twice a day for 3 days. Referred hyperalgesia in the lower abdomen was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load or chemicals was evaluated by counting nociceptive behaviors. Spinal phosphorylated ERK was detected by immunohistochemistry. Cav3.2 knockdown was accomplished by intrathecal injection of antisense oligodeoxynucleotides. Butyrate treatment caused referred hyperalgesia and colonic hypersensitivity to distension in ddY mice, which was abolished by T-channel blockers and/or Cav3.2 knockdown. Butyrate also increased the number of spinal phosphorylated ERK-positive neurons following colonic distension in the anesthetized ddY mice. The butyrate-treated ddY mice also exhibited T-channel-dependent colonic hypersensitivity to intracolonic Na2S, known to enhance Cav3.2 activity, and TRPV1, TRPA1 or proteinase-activated receptor 2 (PAR2) agonists. Wild-type, but not Cav3.2-knockout, mice of a C57BL/6J background, after treated with butyrate, mimicked the T-channel-dependent referred hyperalgesia and colonic hypersensitivity in butyrate-treated ddY mice. Our study provides definitive evidence for an essential role of Cav3.2 in the butyrate-induced colonic pain and nociceptor hypersensitivity, which might serve as a target for treatment of visceral pain in IBS patients.

Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice.

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.

Environmental investigation of potentially pathogenic Vibrio parahaemolyticus in the Seto-Inland Sea, Japan.

Seawater and organic material (live and/or dead matter deposited on any substratum submersed in seawater) were collected during the cool weather season from a coast of the Seto-Inland Sea, Japan, and analyzed to determine Vibrio parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by the polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 95% of the samples were positive for V. parahaemolyticus (with densities of 3 to >1400 cells per 100 ml water or 10 g organic samples) by the most-probable-number (MPN)-PCR technique with species-specific toxR primers, but only 40% were positive by the conventional MPN-culture technique (with densities ranging from 3 to 240 cells per 100 ml water or 10 g organics). Furthermore, the tdh and trh genes were positive in 55% and 20% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN-culture procedure. The difference in detection between the MPN-culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.

Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds.

Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS. When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.

Histamine-releasing reaction induced by the N-terminal domain of Vibrio vulnificus metalloprotease.

A zinc metalloprotease secreted by Vibrio vulnificus, an opportunistic human pathogen causing septicemia and wound infection, stimulates exocytotic histamine release from rat mast cells. This protease consists of two functional domains: the N-terminal domain that catalyzes proteolytic reaction and the C-terminal domain that promotes the association with a protein substrate or cell membrane. Like the intact protease, the N-terminal domain alone also induced histamine release from rat peritoneal mast cells in a dose- and time-dependent manner. However, the reaction induced was apparently weak and went on more slowly. The nickel-substituted protease or its N-terminal domain, each of which has the reduced proteolytic activity due to decreased affinity to a substrate, showed much less histamine-releasing activity. When injected into the rat dorsal skin, the N-terminal domain also evoked enhancement of the hypodermic vascular permeability, while the activity was comparable to that of the protease. Taken together, the protease may stimulate histamine release through the action of the catalytic center of the N-terminal domain on the target substance(s) on the mast cell membrane. The C-terminal domain may support the in vitro action of the N-terminal domain by coordination of the association of the protease with the membrane, but it may not modulate the in vivo action.

Proteolytic activation of Vibrio mimicus (Vm) major outer membrane protein haemagglutinin (HA) with Vm-HA/protease: Implication for understanding bacterial adherence.

Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.