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.

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.

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.

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.

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.

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.

Corneal amyloidosis complicated by trichiasis. Immunohistochemical identification of the amyloid light chain protein

Purpose: To identify the amyloid protein of the corneal amyloidosis complicated by trichiasis.Methods: The two patients were 41-year-old and 38-year-old women with trichiasis. They had gelatinous drop-like corneal change in the hemilateral eye. The lesion was excised and examined by light and electron microscopy. Additionally, we performed an immunohistochemical study with immunofluorescence techniques using cryosections.Results: The amyloid deposits were confirmed with light and electron microscopy. Congo red positive staining was not reduced following pretreatment with potassium permanganate. Immunohistochemically, amyloid deposits in the cornea stained positively with serum human light chain kappa and lambda. Pretreatment of the section with 0.05% Tween-20 did not decrease the staining with fluorescence. The deposits stained negatively with serum prealbumin and keratin antibodies.Conclusions: These findings indicate the protein of the corneal amyloidosis complicating trichiasis to be an amyloid light chain (AL) protein that has never been identified in this kind of corneal amyloidosis.

Limitations in the medical treatment of cryptogenic or symptomatic localization-related epilepsies of childhood onset.

We retrospectively examined 169 patients who had cryptogenic or symptomatic localization-related epilepsies (LRE) and were followed-up for more than 5 years. The probability of seizure control was 0.13 during the first year of treatment, 0.25 during the first 5 years, and 0.09 during the second and third 5 years. No patients who continued to have intractable seizures for 15 years became free of seizures. The onset of LRE at the age of 3 years or less, seizure cluster, mesial temporal sclerosis (MTS), and temporal lobe epilepsy (TLE) were significantly associated with a poor seizure control. If an antiepileptic drug (AED) failed to control seizures, probability of seizure control by the next drug was low, in particular in patients in whom more than 4 AEDs have already been tried, and seizure control could not be expected after a trial of 6 AEDs. A tentative indication of epilepsy surgery for LRE of childhood onset may be 5 years of poor seizure control and/or failure of four AEDs.

Epileptic spasms preceded by partial seizures with a close temporal association.

PURPOSE: To investigate the distinctive features of patients with West syndrome who had partial seizures followed by epileptic spasms (PS-ES). METHODS: We examined 45 patients with West syndrome whose epileptic spasms were recorded with simultaneous video-electroencephalography (EEG) monitoring between 1982 and 1996. We investigated the patients who had PS-ES and compared the PS-ES patients with the 37 patients without PS-ES. RESULTS: Of the 45 patients who had epileptic spasms in clusters (ES) and hypsarrhythmia on the interictal EEG, eight (17%) had ES preceded by partial seizures (PS) with a close temporal association. Seven of these eight were female patients. The underlying disorders were tuberous sclerosis (one patient), Aicardi syndrome (one), nonketotic hyperglycinemia (one), and focal cortical dysplasia (one). The etiology was unknown in the remaining four patients, but was suspected to be of prenatal origin in three. Three types of seizure sequence were identified: PS followed several seconds later by ES (two patients), alternating PS and ES starting with PS (three), and PS gradually replaced by ES with overlapping of the two (three). PS-ES disappeared or was replaced by other types of seizures in 1-34 months. Six patients could not walk, and all patients could not speak any sentences at age 3 years. CONCLUSIONS: Compared with patients without PS-ES, those with PS-ES more often had organic brain lesions of prenatal origin, other types of seizures before the onset of ES, asymmetric hypsarrhythmia on the EEG, and poor psychomotor outcome.

[Corneal amyloidosis complicated by trichiasis--immunohistochemical identification of the amyloid light chain protein].

PURPOSE: To identify the amyloid protein of the corneal amyloidosis complicated by trichiasis. METHODS: The two patients were 41-year-old and 38-year-old women with trichiasis. They had gelatinous drop-like corneal change in hemilateral eye. The lesion was excised and examined by light and electron microscopy. Additionally, we performed an immunohistochemical study with immunofluorescence techniques using cryosections. RESULTS: The amyloid deposits were confirmed with light and electron microscopy. Congo red positive staining was not reduced following pretreatment with potassium permanganate. Immunohistochemically, amyloid deposits in the cornea stained positively with serum human light chain kappa and lambda. Pretreatment of the section with 0.05% Tween-20 did not decrease the staining with fluorescence. The deposits stained negatively with serum prealbumin and keratin antibodies. CONCLUSIONS: These findings indicate the protein of the corneal amyloidosis complicating trichiasis to be an amyloid light chain (AL) protein that has never been identified in this kind of corneal amyloidosis.

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease.

Meningitis is not a common complication of chronic granulomatous disease (CGD). Here, we present details of a 3-year-old boy with X-linked CGD, who suffered from fungal meningitis. While 19 samplings using conventional cerebrospinal fluid (CSF) cultures failed to detect any organisms, fungal DNA was identified in the CSF by a new polymerase chain reaction (PCR)-based method. The patient recovered without any sequelae after treatment with a combination of antifungal agents, interferon-gamma and granulocyte infusions. This case report demonstrates that fungal meningitis must be included in the differential diagnosis of infections in CGD patients and that the PCR-based detection of fungal DNA is a powerful tool for diagnosis.

[Neuronal migration disorders and epilepsy].

We retrospectively studied 32 patients who had cortical dysplasia and epilepsy. Cortical dysplasia was classified into diffuse cortical dysplasia (8 patients), bilateral localized cortical dysplasia (5), unilateral diffuse cortical dysplasia (2), and focal cortical dysplasia (17). The onset of epilepsy was younger in patients with more widespread lesions. At the onset, patients with bilateral lesions generally had symptomatic generalized epilepsy, while those with unilateral cortical dysplasia tended to have symptomatic localization-related epilepsy. In patients with focal cortical dysplasia, however, 4 patients had West syndrome, either at the onset of epilepsy or during the follow up period. Seizure outcome was poor in any type of cortical dysplasia. An evaluation of prognostic factors in patients with focal cortical dysplasia did not show any of statistical significance, including gender, age at onset of epilepsy, psychomotor delay and the presence of high intensity areas in T2-weighted MRI.

[Usefulness of continuous arterial infusion chemotherapy for post operative multiple recurrence and residual hepatocellular carcinoma].

Twenty five patients with postoperative multiple recurrence and residual hepatocellular carcinoma received continuous arterial infusion of CDDP and 5-FU via implanted reservoir. For the next five days, 10 mg/body of CDDP and 250 mg/body of 5-FU using arterial infusion were administered. It was discontinued for two days as one course, and 4 courses were basally administered. The efficacy rate was 60%, and there were 7 (28%) CR (complete response) cases. The survival rate was 76.0% for 1 year and 36.5% for 3 years, which is a favorable result considering their advanced stage. Thus, this treatment seemed to be effective for multiple hepatocellular carcinoma.

Chilblain lupus erythematosus of Hutchinson responding to surgical treatment: a report of two patients with anti-Ro/SS-A antibodies.

We report two patients with chilblain lupus erythematosus of Hutchinson (CL) who responded to surgical treatment. One of them was a 72-year-old woman (case 1), and the other a 62-year-old man (case 2). We attempted to treat these patients by excising the lesions and subsequently performing full-thickness free skin grafting, using skin from the abdominal region. No recurrence was seen in the operated area 7 years (case 1) and 3 years (case 2) after surgery. However, lesions persisted in the areas not operated upon, and in the areas where lesions had not been adequately excised. These results suggest that surgical removal of local factors reduces the rash in these cases. In addition, both patients were serologically positive for the anti-Ro/SS-A antibody suggesting that local expression of the Ro/SS-A antigen may be involved in the pathogenesis of the skin lesions. To our knowledge, full thickness free skin grafting has not been used previously to treat CL-associated skin lesions, and is promising as a treatment for patients who do not respond to conventional means.

[The study of continuous arterial infusion chemotherapy with CDDP and 5-FU in patients with hepatocellular carcinoma].

Between Feb. 1992 and April in 1995, 22 patients with hepatocellular carcinoma (10, recurrence; 12, unresectable) received continuous arterial infusion of CDDP and 5-FU via implanted reservoir. For the next five days, 10 mg/body of CDDP and 250 mg/body of 5-FU using arterial infusion were administered. It was discontinued for two days as one course, and 4 courses were basally administered. Patients were divided into 2 groups (6 hours group and 24 hours group) according to the duration of the administration of 5-FU. There were no differences in efficacy rate between the 2 groups (6 hours group, 64%; 24 hours group, 62.5%), but CR (complete response) cases appeared in only the 6 hours group. There were no severe side effects in the 2 groups, but systemic side effects appeared in 6 hours group more often than in 24 hours group. Only in the 24 hours group, 2 patients had the narrowing and obstruction of hepatic artery which was suggested to be caused by intimal injury due to continuous administration of 5-FU. Continuous arterial infusion chemotherapy with CDDP and 5-FU seemed to be effective. Further study on adequate time and volume of administration including pharmacokinetics is needed to enhance the clinical effectiveness of continuous arterial infusion of CDDP and 5-FU.