Time course and localization of nerve growth factor expression and sensory nerve growth during progression of knee osteoarthritis in rats.

OBJECTIVES: Nerve growth factor (NGF) and sensory nerves are key factors in established osteoarthritis (OA) knee pain. We investigated the time course of NGF expression and sensory nerve growth across early and late stages of OA progression in rat knees. DESIGN: Knee OA was induced by medial meniscectomy in rats. OA histopathology, NGF expression, and calcitonin gene-related peptide immunoreactive (CGRP-IR) nerves were quantified pre-surgery and post-surgery at weeks 1, 2, 4 and 6. Pain-related behavior was evaluated using dynamic weight distribution and mechanical sensitivity of the hind paw. RESULTS: NGF expression in chondrocytes increased from week 1 and remained elevated until the advanced stage. In synovium, NGF expression increased only in early stages, whereas in osteochondral channels and bone marrow, NGF expression increased in the later stages of OA progression. CGRP-IR nerve density in suprapatellar pouch peaked at week 4 and decreased at week 6, whereas in osteochondral channels and bone marrow, CGRP-IR innervation increased through week 6. Percent ipsilateral weight-bearing decreased throughout the OA time course, whereas reduced paw withdrawal thresholds were observed only in later stages. CONCLUSION: During progression of knee OA, time-dependent alterations of NGF expression and CGRP-IR sensory innervation are knee tissue specific. NGF expression increased in early stages and decreased in advanced stage in the synovium but continued to increase in osteochondral channels and bone marrow. Increases in CGRP- IR sensory innervation followed increases in NGF expression, implicating that NGF is a key driver of articular nerve growth associated with OA pain.

Contribution of nerves within osteochondral channels to osteoarthritis knee pain in humans and rats.

OBJECTIVES: Subchondral bone may contribute to knee osteoarthritis (OA) pain. Nerve growth factor (NGF) can stimulate nerve growth through TrkA. We aimed to identify how sensory nerve growth at the osteochondral junction in human and rat knees associates with OA pain. METHODS: Eleven symptomatic chondropathy cases were selected from people undergoing total knee replacement for OA. Twelve asymptomatic chondropathy cases who had not presented with knee pain were selected post-mortem. OA was induced in rat knees by meniscal transection (MNX) and sham-operated rats were used as controls. Twice-daily oral doses (30 mg/kg) of TrkA inhibitor (AR786) or vehicle were administered from before and up to 28 days after OA induction. Joints were analysed for macroscopic appearances of articular surfaces, OA histopathology and calcitonin gene-related peptide-immunoreactive (CGRP-IR) sensory nerves in medial tibial plateaux, and rats were assessed for pain behaviors. RESULTS: The percentage of osteochondral channels containing CGRP-IR nerves in symptomatic chondropathy was higher than in asymptomatic chondropathy (difference: 2.5% [95% CI: 1.1-3.7]), and in MNX-than in sham-operated rat knees (difference: 7.8% [95%CI: 1.7-15.0]). Osteochondral CGRP-IR innervation was significantly associated with pain behavior in rats. Treatment with AR786 prevented the increase in CGRP-IR nerves in osteochondral channels and reduced pain behavior in MNX-operated rats. Structural OA was not significantly affected by AR786 treatment. CONCLUSIONS: CGRP-IR sensory nerves within osteochondral channels are associated with pain in human and rat knee OA. Reduced pathological innervation of the osteochondral junction might contribute to analgesic effects of reduced NGF activity achieved by blocking TrkA.

Impact of medial versus lateral knee pain on deep tissue hyperalgesia and muscle strength.

BACKGROUND: Accumulating evidence indicates that knee pain gives rise to sensory and motor alterations, however, whether different profile of knee pain causes different alterations has not been investigated. The purpose of this experimental study is to clarify characteristics of medial and lateral knee pain and its potential for modulating sensory and motor function in humans. METHODS: Fourteen healthy men were included. Medial knee pain (MP) was induced by injection of hypertonic saline (0.5 mL) into the tibial insertion of the medial collateral ligament. For comparison, lateral knee pain (LP) was induced by injection of hypertonic saline identically into the iliotibial tract. Isotonic saline was injected contralaterally as control. Pain intensity was assessed on a continuous electronic visual analogue scale (VAS). Before, during and after the painful state, pressure pain thresholds from the knee (PPTs), maximal isometric muscle strength of the quadriceps and grip power were assessed bilaterally. RESULTS: MP demonstrated significantly higher VAS scores than LP and compared with control. PPTs decreased on medial and lateral knee in MP but only on the lateral knee in LP. Quadriceps muscle strength and grip power reduced bilaterally in both models, however, MP caused significantly greater reduction of ipsilateral quadriceps strength compared with LP. CONCLUSION: Medial knee pain has a greater impact on deep tissue hyperalgesia and reduction of the muscle strength compared with lateral knee pain. This is a novel finding that should be taken into consideration in a treatment strategy for painful knee patients. SIGNIFICANCE: The experimental medial knee pain model demonstrated higher pain intensity, more localized pain distribution, widespread deep tissue hyperalgesia and more severe inhibition of muscle strength compared with the lateral knee pain model.

Nociceptive phenotype alterations of dorsal root ganglia neurons innervating the subchondral bone in osteoarthritic rat knee joints.

OBJECTIVE: Subchondral bone plays a role in generating knee joint pain in osteoarthritis (OA). The objective of this study was to clarify nociceptive phenotype alterations of subchondral bone afferents of the distal femur in mono-iodoacetate (MIA)-induced OA rats. METHODS: OA was induced by intra-articular injection of MIA in rats. Two different retrograde tracers were separately injected into the knee joint cavity and the subchondral bone to identify joint and subchondral bone afferents. Immunohistochemistry was used at 2 weeks (early stage) and 6 weeks (advanced stage) after MIA injection to determine the expression of nociceptive markers (calcitonin gene-related peptide (CGRP) and tyrosine receptor kinase A (TrkA)) and the soma size distribution of CGRP-immunoreactive (IR) neurons. Histological subchondral bone and cartilage damage was scored according to the Osteoarthritis Research Society International grading system. Pain-related behavior was evaluated using weight distribution and mechanical sensitivity of the hind paw. RESULTS: OA caused an up-regulation of CGRP, TrkA and enlargement of soma size of CGRP-IR neurons in both joint and subchondral bone afferents. CGRP and TrkA expression in subchondral bone afferents gradually increased over 6 weeks. Furthermore, up-regulation of CGRP and TrkA in subchondral bone afferents displayed a strong correlation with the subchondral bone damage score. CONCLUSION: Up-regulation of nociceptive markers in subchondral bone afferents correlated with subchondral bone damage, suggesting that subchondral bone is a therapeutic target, especially in the case of advanced stage knee OA. In particular, CGRP and TrkA are potentially molecular therapeutic targets to treat joint pain associated with subchondral lesions.

Repeated intra-articular injections of acidic saline produce long-lasting joint pain and widespread hyperalgesia.

BACKGROUND: Synovial fluid in inflamed joint shows a drop in pH, which activates proton-gated ion channels in nociceptors. No studies have ever tried to develop and characterize acid-induced joint pain. METHODS: Rats were injected intra-articularly with pH 4.0 acidic saline twice, 5 days apart. Pain-related behaviour tests including weight-bearing asymmetry, paw withdrawal threshold and knee compression threshold were conducted. To clarify the roles of proton-gated ion channels, rats were injected intra-articularly with selective antagonists for ASIC1a, ASIC3 and TRPV1 on day 5 (before the second injection) or on day 14. Underlying peripheral and central pain mechanisms were evaluated using joint histology, interleukin-1ß concentrations in the synovium, single-fibre recording of the knee afferent and expression of phosphorylated cyclic adenosine monophosphate-responsive element-binding protein (p-CREB) in the spinal dorsal horn. RESULTS: Repeated injections of acidic saline induced weight-bearing asymmetry, decrease in paw withdrawal threshold and knee compression threshold bilaterally, which lasted until day 28. Early administration of ASIC3 antagonist reduced the bilateral and long-lasting hyperalgesia. Neither articular degeneration nor synovial inflammation was observed. C-fibre of the knee afferent was activated by acidic saline, which was attenuated by pre-injection of ASIC3 antagonist. p-CREB expression was transiently up-regulated bilaterally on day 6, but not on day 14. CONCLUSION: We developed and characterized a model of acid-induced long-lasting bilateral joint pain. Peripheral ASIC3 and spinal p-CREB played important roles for the development of hyperalgesia. This animal model gives insights into the mechanisms of joint pain, which is helpful in developing better pain treatments.

Effects of intra-articular hyaluronic acid injection on immunohistochemical characterization of joint afferents in a rat model of knee osteoarthritis.

BACKGROUND: Intra-articular hyaluronic acid (HA) injection, known as viscosupplementation, is a widely used therapy for pain relief in knee osteoarthritis (OA). Long-term clinical efficacy of HA has been reported in spite of a relatively short residence time. Herein, we evaluated our hypothesis that intra-articular HA injection could reduce the OA-associated changes in joint afferents. METHODS: OA was induced by intra-articular injection of mono-iodoacetate in rats. Animals in the OA + HA group were given three weekly intra-articular HA injections. Pain-related behaviours, including weight-bearing asymmetry and mechanical hyperalgesia of the paw, knee joint histology and immunohistochemistry of joint afferents identified by retrograde labelling, were compared between groups (naïve, OA and OA + HA). RESULTS: OA rats showed pain-related behaviours and up-regulation of pain-related neurochemical markers [calcitonin gene-related peptide (CGRP), tyrosine receptor kinase A (TrkA) and acid-sensing ion channel 3 (ASIC3)] in joint afferents. HA injections reduced not only the severity of OA and pain behaviours but also OA-associated neurochemical changes in joint afferents. The differences between OA and OA + HA were statistically significant in CGRP (61 ± 10% vs. 51 ± 10%; p = 0.0406) but not significant in TrkA (62 ± 10% vs. 54 ± 9%; p = 0.0878) and ASIC3 (38 ± 9% vs. 32 ± 8%; p = 0.3681). CONCLUSION: Intra-articular HA injections reduced the severity of OA, decreased mechanical hyperalgesia of the paw, but not weight-bearing asymmetry, and attenuated OA-associated up-regulation of CGRP, but not TrkA and ASIC3, in joint afferents. The modulatory effects of HA on joint afferents is one of the underlying mechanisms of the gap between HA residence time and duration of clinical efficacy.

PET in infancy predicts long-term outcome during adolescence in cryptogenic West syndrome.

BACKGROUND AND PURPOSE: Developmental and seizure outcomes in patients with cryptogenic West syndrome are variable. Our aim was to clarify the relationship between FDG-PET findings in infancy and long-term seizure and developmental outcome in cryptogenic West syndrome. MATERIALS AND METHODS: From 1991 to 1999, we prospectively performed FDG-PET from the onset of cryptogenic West syndrome in 27 patients. PET was performed at onset and at 10 months of age. In 2012, we evaluated the educational status, psychomotor development, and seizure outcome in 23 of the 27 patients (13-22 years of age). The correlation between PET findings and outcome was evaluated. RESULTS: At onset, PET showed hypometabolism in 13 patients (57%). The second PET after the initial treatment revealed cortical hypometabolism in 7 patients (30%). While hypometabolism at onset disappeared on the second PET in 9 patients, it was newly revealed in 3 patients on the second PET. In 2012, seven patients had persistent or recurrent seizures. Eight patients had intellectual impairment. The first PET did not correlate with seizure or developmental outcome. Five of 7 patients (71%) with hypometabolism seen on the second PET had persistent or recurrent seizures, while 14 of 16 (88%) patients with normal findings on the second PET were free of seizures. Five of 7 patients (71%) showing hypometabolism on the second PET had intellectual impairment. Thirteen of 16 (81%) patients with normal findings on the second PET showed normal intelligence. A significant correlation was found between the second PET and long-term seizure (P = .01) or developmental outcome (P = .03). CONCLUSIONS: Cortical hypometabolism is not permanent; it changes with clinical symptoms. Hypometabolism after initial treatment predicts long-term seizures and poor developmental outcome.

Nociceptive phenotype of dorsal root ganglia neurons innervating the subchondral bone in rat knee joints.

BACKGROUND: The subchondral bone of the distal femur is a source of pain caused by osteoarthritis (OA) or spontaneous osteonecrosis of the knee. However, nociceptive phenotype of dorsal root ganglia (DRG) neurons innervating the subchondral bone in rat knee joints has not been clarified. METHODS: Retrograde labelling was used to identify afferents innervating the subchondral bone of the distal femur and the knee joint in rats. The nociceptive phenotype markers [calcitonin gene-related peptide (CGRP), tyrosine receptor kinase A (TrkA), neurofilament 200 (NF200) and isolectin B4 (IB4)], segmental distribution and the soma size of backlabelled DRG neurons were examined. Furthermore, we evaluated the differences in nociceptive phenotype between the subchondral bone and the knee joint afferents. RESULTS: The majority (60%) of the subchondral bone afferents were localized in L3 DRGs and fewer in L4 and L5, while the knee joint afferents were localized mainly in L3 and L4. The percentage of CGRP immunoreactive (IR), TrkA-IR, NF200-IR and IB4-binding neurons in the subchondral bone afferents were 50%, 65%, 35% and 0%, respectively. The percentage of CGRP-IR and TrkA-IR neurons in the subchondral bone afferents was significantly higher than that in the knee joint afferents, respectively (p < 0.05). CONCLUSION: The majority of sensory DRG neurons innervating the subchondral bone of the distal femur were CGRP-IR and TrkA-IR. It is expected that therapeutic approaches targeting CGRP and TrkA could be effective in attenuating pain from the subchondral bone in knee joints.

Dependence of fructooligosaccharide content on activity of fructooligosaccharide-metabolizing enzymes in yacon (Smallanthus sonchifolius) tuberous roots during storage.

Tuberous roots of yacon (Smallanthus sonchifolius) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy benefits. However, we have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting even under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8 degrees C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (beta-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on sucrose, both invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FEH acting on short FOSs such as 1-kestose (GF(2)) and 1-nystose (GF(3)) was higher than that of 1-FFT. These results suggest that the continuous decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand, FOSs with a DP of >or= 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct 1-FEH activity was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connected with the 1-FEH activity.

Macrophage inflammatory protein-2 induced by TNF-alpha plays a pivotal role in concanavalin A-induced liver injury in mice.

BACKGROUND/AIMS: Macrophage inflammatory protein-2 (MIP-2), one of the CXC chemokines, is involved in the recruitment of neutrophils in several tissue injuries. In this study, we investigated the role of MIP-2 in concanavalin A (Con A)-induced liver injury in mice. METHODS: Liver injury was induced by intravenous injection of Con A (15 mg/kg) and plasma alanine aminotransferase (ALT), MIP-2 levels were determined and histological assessment of the liver was performed. Anti-mouse MIP-2 antibody was intravenously administered 30 min before Con A injection. RESULTS: The plasma ALT level significantly elevated and reached a maximum at 8 h after Con A injection. The plasma MIP-2 level was also elevated and reached a peak value at 2 h after Con A injection. The elevated ALT level by Con A injection was significantly inhibited by the MIP-2 antibody. The elevated plasma MIP-2 level after Con A injection was significantly reduced by the tumor necrosis factor alpha (TNF-alpha) antibody, and MIP-2 was induced in plasma after recombinant TNF-alpha injection. Hepatic necrosis and infiltration of neutrophils were observed after Con A injection, and these histological changes were attenuated by the MIP-2 antibody. CONCLUSIONS: These findings suggest that Con A induces TNF-alpha release, and this TNF-alpha stimulates MIP-2 induction, at least partially contributing to the liver injury mediated through the recruitment of neutrophils.

Pyrrolo[2,3-d]pyrimidine thymidylate synthase inhibitors: design and synthesis of one-carbon bridge derivatives.

A series of novel pyrrolo[2,3-d]pyrimidine derivatives was designed and synthesized as thymidylate synthase (TS) inhibitors. Molecular design was performed on the human TS complex model built on the basis of the reported structure of TS-deoxyuridinemonophosphate (dUMP)-CB3717 ternary complex. From a docking study, we expected that a one-carbon bridge between pyrrolo[2,3-d]pyrimidine and an aromatic ring was suitable. Moreover, we found that the bridge carbon could be replaced with an alkyl group to fill out the unoccupied space. Based on this design, we synthesized five pyrrolo[2,3-d]pyrimidine derivatives with one-carbon bridge and evaluated their TS inhibitory activities. All synthesized compounds inhibited TS more potently than compound 2 (LY231514), and the C8-ethyl analogue (7) showed a remarkable inhibitory activity against TS (IC50=0.017 microM).

Cause of death in renal transplant patients: a comparison between azathioprine and ciclosporin.

The results of renal transplantation have improved due to advances in immunosuppression techniques of preservation, and pre- and postoperative treatments; however, both morbidity and mortality remain serious problems. To decrease the morbidity and mortality rates we analyzed the causes of death after renal transplantation in our hospital. Between 1972 and 1999, we performed 364 renal transplantations, 257 of which were living-related and 107, cadaveric. There were 178 patients given azathioprine and 186 given ciclosporin. The survival rate of the patients on ciclosporin therapy was much better than that of those on azathioprine therapy. Of the total 364 renal transplant patients, 59 (16.2%) died, and 28 (47.5%) of these 59 deaths occurred within 1 year after renal transplantation. The causes of death were infection in 19 (32.2%) patients, gastrointestinal diseases in 16 (27.1%), cardiovascular diseases in 11 (18.6%), cerebrovascular diseases in 6 (10.2%), suicide in 3 (5.1%), and other causes in 4 (6.8%). These findings reinforce that early diagnosis and treatment are essential to decrease the morbidity and mortality rates assoiated with renal transplantation.

Free radical-mediated tolbutamide desensitization of K+ATP channels in rat pancreatic beta-cells.

To study the effects of hydroxyl radicals on the sensitivity of the ATP-sensitive K+ (K+ ATP) channel to tolbutamide, we used patch clamp and microfluorometric techniques in pancreatic beta-cells isolated from rats. cell-attached membrane patches, exposure of the cells to 0.3 mM H2O2 increased the probability of opening of K+ATP channels in the presence of 2.8 mM glucose. Tolbutamide dose-dependently inhibited the K+ATP channel with half-maximal inhibition (IC50) at 0.8 microM before and immediately after exposure to H2O2. After prolonged exposure (>20 min) to H2O2, the IC50 was increased to 15 microM. The presence of both ATP and ADP at concentrations ranging from 0.01 to 0.1 mM in the inside-out bath solution significantly enhanced the inhibition of the channels by 10 microM tolbutamide. Addition of 0.3 mM H2O2 induced a transient minute increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) within 10 min, followed by a sustained pronounced increase in [Ca2]i. After more than 20 min of exposure of cells to 0.3mM H2O2, [Ca2]i was increased to above 2 microM. Treatment of the cytoplasmic face of inside-out membrane patches with 1 microM Ca2+ attenuated the tolbutamide-sensitivity of the K+ATP channel, but not the ATP-sensitivity of the channel. These findings indicate that H2O2 reduces tolbutamide sensitivity by inducing a sustained increase in [Ca2+]i.

[Evaluation and improvement of medical and nursing service and caregiving for the elderly using MDS. 3. Revised recording system of diagnoses and symptoms suitable for use in Japan].

Diagnoses and symptoms are recorded by physicians in the chapter E of the Minimum Data Set (MDS), if they are relevant to disabilities of activities of daily living, cognition, behavior, medical treatments or risk of death. We improved the chapter so that it is suited to disease patterns in Japan in a format useful not only for nursing and ADL care but for medical treatment in our practice. In E1, diseases directly underlying the current disability states were recorded in the international classification of Disease, 9th Revision (ICD9). In 24, 670, 195, and 45 patients respectively, there were 0, 1, 2, and 3 separate recorded diseases. A Total of 63 ICD9 codes were observed, but only four codes; 290, 332, 431, and 434, were underlying diseases for 3% or more patients. These codes included mostly ischemic and degenerative disease of the brain.

Transient seizure remission in intractable localization-related epilepsy.

We sought to elucidate the clinical features of transient seizure remission in intractable cryptogenic or symptomatic localization-related epilepsy of childhood onset. Transient seizure remission has been reported to occur in mesial temporal sclerosis or focal cortical dysplasia, but few reports have focused on this phenomenon. We retrospectively scrutinized the temporal profiles of seizure frequency of 99 patients with intractable localization-related epilepsy by reviewing their medical charts. Ten patients (10%) had transient seizure remissions that lasted for 2 years or longer. When an appropriate antiepileptic agent was administered, seizure remission occurred within 1-18 months. Without any triggering factors, the seizures recurred abruptly in seven patients and gradually in three. Epileptiform discharges on electroencephalography disappeared during the transient remission in seven patients and reappeared in five of them after recurrence. After recurrence, no antiepileptic agent was able to control the seizures. In comparison with those without transient seizure remission, these 10 patients tended to have normal intelligence and a positive family history for epilepsy. Transient seizure remission occurs in a variety of pathologic changes and may be a result of an interaction between the progressive nature of some types of epileptogenic foci and an effect of the antiepileptic drugs.