Preparation and first biological evaluation of novel Re-188/Tc-99m peptide conjugates with substance-P

IntroductionNew ¹⁸⁸Re and ^99mTc peptide conjugates with substance- P (SP) were prepared and biologically evaluated. The radiopharmaceuticals have been labelled with the [M≡N]²⁺ (M=^99mTc, ¹⁸⁸Re) core using a combination of π-donor tridentate and π-acceptor monodentate ancillary ligands.MethodsThe new radiopharmaceuticals have been prepared through a two-step reaction by simultaneous addition of the tridentate and monodentate ligands to a vial containing a preformed [M≡N]²⁺ core. The tridentate ligand was formed by linking two cysteine residues to the terminal arginine of the undecapeptide SP, whereas the monodentate ligand was a tertiary phosphine. The preparation of the corresponding Re-188 derivative required developing a more complex chemical procedure to obtain the [Re≡N]²⁺ core in satisfactory yields. Characterization of the resulting products was obtained by chromatographic methods. Biological evaluation was performed for both Tc-99m and Re-188 derivatives by in-vitro studies on isolated cells expressing NK1-receptors. In-vivo imaging in mice was carried out using a small-animal YAP(S)PET tomograph.ConclusionNew Tc-99m and Re-188 peptide radiopharmaceuticals with SP have been prepared in high-yield and with high-specific activity. Both Tc-99m and Re-188 peptide radioconjugates exhibit high affinity for NK1 receptors, thus giving further evidence to the empirical rule that structurally related Tc-99m and Re-188 radiopharmaceuticals exhibit identical biological properties.     

Distribution of substance-P nerves inside the infrapatellar fat pad and the adjacent synovial tissue: a neurohistological approach to anterior knee pain syndrome

Introduction The purpose of the study was to determine the distribution and number of nerves inside the infrapatellar fat pad and the adjacent synovium, in particular with regards to nociceptive substance-P nerves.Materials and methods The infrapatellar fat pad of the knee was resected from 21 patients (4 male, 17 female, mean age 69 years) during the course of standard total knee arthroplasty operations performed in our clinic. The fat pad was dissected into five standardized segments, fixed in formalin and embedded in paraffin. Immunohistochemical techniques using antibodies against S-100 protein and substance-P (SP) were employed to determine and specify the nerves.Results Studying all the detectable nerves present in 50 observation fields (200-fold magnification), we found an average of 106 S-100 versus 25 SP nerves (24%) in the synovium and 27 S-100- versus 7 SP nerves (26%) in the interior of the fat pad. The total nerve count was significantly ( P <0.001) higher in the synovium than in the fat pad for both marker types. The number of S-100 nerves was significantly ( P <0.05) higher in the central and lateral segments of the fat pad, while SP nerves were equally distributed throughout all segments of the fat-pad. SP nerves were significantly more frequently associated with blood vessels inside the fat pad (43%, P <0.05) than in the synovial tissue (28%).Conclusion The occurrence and distribution of SP nerves inside the infrapatellar fat pad suggest a nociceptive function and a neurohistological role in anterior knee pain syndrome. The data support the hypothesis that a neurogenous infection of the infrapatellar fat pad could contribute to anterior knee pain syndrome.     

Distribution of substance-P nerve fibers in the knee joint in patients with anterior knee pain syndrome A preliminary report

The etiology of pain in anterior knee pain syndrome is a matter of controversy. The normal, articular cartilage is aneural, so defects in the surface are not thought to produce pain. Some authors have sought the origin of the pain in soft tissue structures around the knee. Knowledge of the distribution of nociceptive nerve fibers around the knee would provide insight for treating anterior knee pain syndrome. Twenty consecutive patients (28 knees), all women, with anterior knee pain syndrome (group I) participated in the study. For comparison we used two groups of patients: 20 patients with an osteoarthritic knee (group II) and 20 patients with anterior cruciate ligament rupture or meniscal lesion with no history of pain in the anterior compartment (group III). Immunohistochemical techniques using a monoclonal antibody to substance-P (SP) were employed to identify nociceptive fibers. For statistical analyses we used the one-way ANOVA test, which was corrected with the LSD test, at the level of significance P < 0.05. Results of the study demonstrate that SP-immunoreactive nerve fibers are widespread within the soft tissues around the knee. These tissues include the retinaculum, synovium, fat pad and, in some circumstances, bone. In cases of anterior knee pain, the presence of neuropeptide-containing fibers was statistically significant in the medial retinaculum (P < 0.005) and in the fat pad (P < 0.001) compared to group III, and compared to group II (P < 0.05 and P < 0.007, respectively). For lateral retinaculum this relationship was not so statistically strong (P < 0.02) and was equal in comparison between anterior knee pain patients (group I) and group II or group III. There were no statistically significant differences in the distribution of substance-P nerve fibers in the fat pad, lateral and medial retinaculum or synovium between groups II and III. The results of this study provide immunohistochemical evidence suggesting that pain may originate in the fat pad and medial retinaculum of many patients with anterior knee pain syndrome. Received: 17 December 1997 Accepted: 20 October 1998     

Prothrombotic roles of substance-P, neurokinin-1 receptors and leukocytes in the platelet-dependent clot formation in whole blood

A number of types of non-neuronal cells including leukocytes have been confirmed to possess substance-P and its specific neurokinin-1 receptor (NK1R), while the pathophysiological roles of substance-P in these cells remain to be established. Effects of substance-P through NK1R on platelet-dependent clot formation were evaluated by using an oscillating-probe viscoelastometer. The clot signal, indicative of the clot strength in blood-derived samples, was measured after the stimulation with celite and Ca²⁺. Substance-P (10 nM) increased the clot signal of whole blood obtained from healthy volunteers, especially modulating the platelet-dependent distinctive peak in traces of the signal. A NK1R antagonist Spantide (500 nM) blocked such substance-P derived change, suggesting the involvement of platelets in the action of substance-P. In contrast, substance-P did not increase the clot signal of platelet-containing but leukocyte-removed plasma. From these, we conclude that substance-P promotes platelet-dependent clot formation through NK1R, in which leukocytes appear to be involved.     

Neuropeptide co-release with GABA may explain functional non-monotonic uncertainty responses in dopamine neurons

Co-release of the inhibitory neurotransmitter GABA and the neuropeptide substance-P (SP) from single axons is a conspicuous feature of the basal ganglia, yet its computational role, if any, has not been resolved. In a new learning model, co-release of GABA and SP from axons of striatal projection neurons emerges as a highly efficient way to compute the uncertainty responses that are exhibited by dopamine (DA) neurons when animals adapt to probabilistic contingencies between rewards and the stimuli that predict their delivery. Such uncertainty-related dopamine release appears to be an adaptive phenotype, because it promotes behavioral switching at opportune times. Understanding the computational linkages between SP and DA in the basal ganglia is important, because Huntington’s disease is characterized by massive SP depletion, whereas Parkinson’s disease is characterized by massive DA depletion.     

大鼠旁巨细胞外侧核的免疫细胞化学特性的研究

用免疫细胞化学ＰＡＰ法研究了５－羟色胺（５－ＨＴ）、Ｐ物质（ＳＰ）、亮氨酸脑啡肽（Ｌ－ＥＮＫ）和神经肽Ｙ（ＮＰＹ）阳性神经元在大鼠旁巨细胞外侧核（ＰＧＣＬ）的分布。结果表明：５－ＨＴ、ＳＰ和Ｌ－ＥＮＫ阳性细胞较多，从ＰＧＣＬ的吻端至尾端呈柱状分布，５－ＨＴ阳性细胞较靠近延髓腹侧表面和内侧，ＳＰ和ＬＥＮＫ阳性细胞分布的范围较广，靠近外侧；ＮＰＹ阳性细胞较少，多集中在ＰＧＣＬ的尾侧至中分的区域。     

Distribution of substance-P nerve fibers in intact and ruptured human anterior cruciate ligament: a semi-quantitative immunohistochemical assessment

Numerous studies have reported qualitative and quantitative analysis of nerve supply in the anterior cruciate ligament; however, as yet relatively little is known about the distribution of substance-P nerve endings in the human anterior cruciate ligament. The objective of this work was to evaluate the distribution of substance-P nerve fibers in intact human anterior cruciate ligament, and determine if rupture of the ligament has any influence on occurrence of these receptors. The intact anterior cruciate ligament group (group 1) of osteoarthritis knee, undergoing total knee arthroplasty, consisted of nine patients (eight females) with a mean age of 65.3 years at surgery. The anterior cruciate ligament rupture group (group 2) consisted of 20 patients (18 males and 2 females) with a mean age of 27.8 years at reconstruction. Healing time of the torn ligament in vivo, determined by the time period between the rupture and reconstruction, lasted from 1 to 40 months and the patients were divided into 3 groups (I, II and III) embracing diverse time periods. All harvested anterior cruciate ligaments were sectioned in thirds so that there was a proximal, middle and distal third for each ligament. The distribution of nociceptive receptors was studied by immunohistochemistry with monoclonal antibody to substance-P, including the semi-quantitative assessment. No significant difference was found between the number of substance-P nerve fibers in the proximal, middle and distal third of the intact anterior cruciate ligament ( p >0.05). During the first 4 months after injury (group I) the mean number of neuropeptide-containing fibers was greater in the proximal than in the distal third ( p =0.048996). The number of SP-positive nerve fibers in the proximal third decreased between 5 and 12 months after rupture, in a statistically significant manner ( p =0.045864). This study showed that distribution of the nociceptive nerve supply, positively stained for substance-P, is equal among the intact anterior cruciate ligament. The substance-P nerve ending density was significantly affected by the injury as well as by the time since rupture. The results of this study provide immunohistochemical evidence suggesting that between 1 to 4 months after rupture the site of the injury undergoes neurogenic inflammation, which could have an influence on the healing course of the torn ligament.     

Nestin immunoreactivity in local neurons of the adult rat striatum after remote cortical injury

Nestin is a marker for the neuronal and glial precursor cells and is expressed in reactive astrocytes after brain injury. Following restricted neocortical injury, we found that cells with neuronal morphology in the adult rat striatum became immunoreactive for both nestin and the neuronal marker, microtubule-associated protein 2 (MAP-2), but not for the astroglial marker, glial fibrillary acidic protein (GFAP). The number of nestin-positive cells transiently increased in the striatum. Continuous administration of 5-bromo-2'-deoxyuridine (BrdU) after cortical injury did not reveal any newly generated neurons in the striatum. Double-labeling fluorescent immunocytochemistry revealed that the nestin-positive striatal cells were also substance-P-positive. These findings suggest that some factors released from the injured cortex may induce nestin immunoreactivity in striatal neurons.     

Gingival crevicular fluid and plasma levels of neuropeptide Substance-P in periodontal health, disease and after nonsurgical therapy

Background and Objective: The level of Substance‐P in gingival crevicular fluid has been found to correlate with clinical measures of periodontal disease. The present study was designed to assess the relationship between clinical parameters and levels of Substance‐P in the gingival crevicular fluid from inflamed gingiva, periodontitis sites and after treatment of periodontitis sites, and to correlate them to the Substance‐P levels of plasma. Material and Methods: Thirty, age‐ and gender‐matched subjects were divided into three groups (healthy, gingivitis and chronic periodontitis) based on modified gingival index scores and clinical attachment loss. A fourth group consisted of 10 subjects from the periodontitis group, 6–8 wk after initial therapy. Plasma and gingival crevicular fluid samples were collected and quantified for Substance‐P using an enzyme immunoassay. Results: The mean concentration of Substance‐P, both in gingival crevicular fluid and plasma, was observed to be highest in the periodontitis group (45.13 pg/mL in gingival crevicular fluid and 67.8 pg/mL in plasma) and lowest in the healthy group (6.07 pg/mL in gingival crevicular fluid and below the detection level in plasma). The mean Substance‐P concentration in the gingivitis group (11.42 pg/mL in gingival crevicular fluid and 38.8 pg/mL in plasma) and in the after‐treatment group (7.58 pg/mL in gingival crevicular fluid and 39.7 pg/mL in plasma) lay between the highest and lowest values. In all groups the gingival crevicular fluid levels showed a statistically significant positive correlation with that of plasma and clinical attachment loss. Conclusion: Substance‐P levels were highest in the gingival crevicular fluid from sites with periodontal destruction; however, periodontal treatment resulted in the reduction of Substance‐P levels. Gingival crevicular fluid and plasma Substance‐P levels showed a positive correlation in all of the groups.