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.

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.

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.

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.

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.

Culture of normal human epidermal cells with 3T3 feeders on millipore filters.

Human epidermal cells were cultured with lethally irradiated 3T3 cells and grew as large colonies both on plastic and millipore filters. Subcultures were possible. The grown colonies were studied morphologically and histologically. The whole intact crossview of epidermal colonies in paraffin sections was obtained from the cell culture on millipore filters. The epidermal cells derived from younger persons had higher efficiency of colony formation than those from older ones. The membrane filter was shown to be good substratum for epidermal cell growth as well as plastic.

Long-term organ culture of rabbit skin: effect of EGF on epidermal structure in vitro.

A method is described for maintaining the epidermal structure of normal rabbit ear skin explants in organ culture for up to 12 weeks. Split-thickness skin specimens were put in diffusion chambers made of either millipore filters or bovine collagen membranes, and then submitted to a roller tube culture at 15 rpm and 36 degrees C. The culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum (FCS) + 0.4 micrograms/ml hydrocortisone. The gas used in the culture tube was air + 5% CO2. Autoradiography revealed the incorporation of [3H]-glycine into the 68-kD keratin band of explants for up to 12 weeks, indicating that normal keratinization was maintained throughout the entire culture period. The turnover time of the epidermis from basal layer to granular layer was around 7 d in both the early and late stages of culture. The addition of epidermal growth factor (EGF) to the culture caused the epidermis to become acanthotic with orthokeratosis, but with high concentrations of EGF (greater than or equal to 10 ng/ml) parakeratosis and increased proliferation of the epidermis occurred. Dexamethasone (DMS) strongly inhibited the EGF effect.

Organ culture of psoriatic lesions: appearance of granular layers in vitamin A-free culture media.

Some morphologic changes of the epidermis of psoriatic skin were observed in organ culture in the presence of absence of vitamin A. Normal and uninvolved and involved psoriatic skin areas, punch biopsied in 3-4-mm diameter specimens, were put in serum-free medium containing no vitamin A with or without delipidized fetal calf serum (FCS) and rotation cultured at 60 rpm under an atmosphere consisting of 95% O2 + 5% CO2. The involved psoriatic skin specimens showed well-developed granular layers after 1 d of culture. The values of labeling indices of 3H-thymidine (3H-TdR) incorporated into the epidermal layers of the cultured specimens of the psoriatic skin were nearly constant during culture for as long as 8 d, although the viable epidermal layer gradually became thinner. Addition of tretinoin to the culture caused uninvolved and involved psoriatic skin specimens to become parakeratotic at concentrations as low as about 2.0 x 10(-6) M and 4.0 x 10(-8) M, respectively, suggesting that the involved psoriatic epidermis is much more sensitive, in terms of keratinization, than uninvolved epidermis to the effect of tretinoin.

Reduced epidermal cyclic AMP accumulation following prostaglandin stimulation: Its possible role in the pathophysiology of psoriasis.

Since prostaglandins are known to be one of the modulators of cyclic AMP, a study of the role of prostaglandins and their relation to the regulation of the nucleotide in psoriasis was initiated. Guinea-pig epidermal preparations were incubated with prostaglandins (PGs) in the presence of theophylline. PGE1 increased cyclic AMP levels 3.5-fold whereas a 2-fold increase was seen with PGE2 and PGA1. The increase in cyclic AMP accumulation was linear with increasing concentration of PGE1, from 10(-11) to 10(-5) M. PGE1 significantly increased cyclic AMP in psoriatic epidermis in vitro; however, the stimulation of cyclic AMP accumulation was significantly less in involved epidermis as compared with uninvolved tissue. The specificity of this stimulation, its occurrence at physiologic levels, and the decreased responsiveness of the cyclic AMP system in psoriatic epidermis to PGE1 stimulation suggest that the altered response of epidermis to PGs may be one of the factors in the pathophysiology of psoriasis.

The role of prostaglandin E, cyclic AMP, and cyclic GMP in the proliferation of guinea-pig ear skin stimulated by topical application of vitamin A acid.

Daily treatment of guinea-pig ear skin with topical 0.5% retinoic acid in acetone produced erythematous scaly dermatitis. Histologic sections revealed bandlike thickening of the epidermis on days 2 to 4, psoriasiform acanthosis, papillomatosis and increased mitotic activity on days 5 to 6. Also seen were dilatation of the upper dermal blood vessels and a fibroblastic, histiocytic reaction in the dermis. Prostaglandin E, cyclic AMP, and cyclic GMP levels were increased in the treated skin and thymidine incorporation was enhanced. Cyclic AMP and GMP levels peaked on day 5 simultaneous with maximal epidermal hyperplasia, increased mitotic activity and dermal reaction. Tritiated thymidine uptake peaked on days 4 and 5, and prostaglandin E levels continued to increase up to day 6. Cyclic AMP phosphodiesterase activity of treated skin on day 5 did not appear to be significantly different from control.

Epidermal synthesis of prostaglandins and their effect on levels of cyclic adenosine 3', 5'monophosphate.

Extracts of guinea-pig and human skin epidermis were analyzed for prostaglandins PGE1, E2, and F2alpha by radioimmunoassy, and found to contain a total of 62.0 (guinea pig) and 144.7 (human) ng/gm wet weight. the three prostaglandins occurred in approximately equal amounts. Guinea=pig epidermal homogenates converted labeled arachidonic acid to PGE2 and PGF2alpha, the rate of formaiton being 10 and 2.5 pmoles per mg protein in O.K hr, respectively. Conversion in the dermis occurred to a much smaller extent. Homogenates of univolved and involved epidermis from 10 subjects with psoriasis produced PGE2 from arachidonic acid at rates of 6.48 and 2...

The presumption of proteolytic enzymes related to the formation of intermediates during the terminal differentiation.

The [14C]Gly-labelled keratin polypeptides extracted with 1% SDS and 10 mM DTT were made to undergo changes with an enzyme fraction (ammonium sulfate, 50-75% saturated fraction) prepared from a human epidermis in the presence of 1% Triton X-100. In particular, 69-67 kDa peptides were considerably decreased with the above enzyme fraction in the time course experiments, and the components strongly bound to the cell membrane had little effect on the above reaction. In addition, in the case of the [14C]Gly-labelled keratin filament assembly, 69 and 62 kDa peptides were decreased and 55, 52 and 50 kDa peptides were increased with the same enzyme fraction in the time course experiments. From these results, we estimated that the proteolytic enzyme(s) may exist in the human epidermis, and may be processed to keratin intermediates from prekeratin during the initial stage of terminal differentiation in the human epidermis.

Intermediates in the conversion of prekeratin into keratin molecules in human epidermis.

In order to elucidate the relationship between prekeratin and keratin, we performed pulse-chase experiments using [35S]methionine (35S-Met) in vitro. Of 6 prekeratin molecules (49, 52, 55, 62, 69 and 71 kDa) that incorporated 35S-Met, the 55-kDa prekeratin incorporated the most 35S-Met. In 3 molecules (52, 55 and 62 kDa) incorporation was decreased at 30 min after being chased; however, incorporation of only two molecules (55 and 62 kDa) of the 6 prekeratins was increased at 60 min. From these results and our previous data, we conclude that the initial stage of processing is as follows: 3 prekeratin molecules (52, 55 and 62 kDa) are first cleaved in the N-terminal region, then two prekeratin molecules (55 and 69 kDa) are processed to intermediates (52 and 62 kDa) by some proteolytic enzyme(s).

Self-induced photogenic epilepsy in infants.

We studied two infants with self-induced photogenic epilepsy and investigated their seizures with simultaneous EEG and videotape recording. A 2-year-old boy showed peculiar head-nodding behavior towards bright light before he manifested myoclonic absence or myoclonic seizures. A 14-month-old infant girl showed blinking in front of the television set before myoclonic jerks developed. Neither head nodding nor blinking was associated with paroxysmal discharges. We concluded that such preictal behavior was not part of the seizure.

Complex partial seizures in children: ictal manifestations and their relation to clinical course.

We analyzed complex partial seizures in 38 children aged 0 to 13 years, using simultaneous EEG-VTR recording. In infants, seizure duration was longer, automatisms were less purposeful and more common in the oral area, and convulsive movements were frequently seen and more extensive in comparison with older children. In ictal EEG, spiky components frequently appeared in infants. Parietal or occipital ictal foci were more often associated with mental or physical abnormalities than were frontal or central foci. Extensive convulsive movements were inversely proportional to seizure prognosis in older children. Interictal paroxysmal discharges were observed less frequently in infants.

Recombinant Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate antimalarials.

We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains. The validity of the screen was verified by the subsequent examination of several substituted pyrrolo[2,3-d]pyrimidines for their antimalarial activity. Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme. As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate. This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308. The compounds inhibited the growth of drug-sensitive cultured P. falciparum with 50% effective concentrations of the ranged 0.17-30 nM. As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500. Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P. berghei in mice.

Relapse of herpes simplex encephalitis in children.

The polymerase chain reaction method was used to diagnose herpes simplex encephalitis in children. Initial samples of cerebrospinal fluid from 15 patients with herpes simplex encephalitis were all positive for the herpes simplex virus DNA by polymerase chain reaction assay. In terms of early diagnosis, polymerase chain reaction assay became positive significantly earlier than the detection of intrathecally produced anti-herpes simplex virus antibody using the enzyme-linked immunosorbent assay (4.4 vs 8.9 days after onset; P less than .01). Serial examinations showed that the presence of virus DNA in cerebrospinal fluid continued for 3 to 18 days after the neurologic onset (mean 10.1 days). Four of the 15 patients had a relapse of encephalitis after completing acyclovir therapy. The mean duration of initial acyclovir therapy in the recurrent group was significantly shorter than that in the nonrecurrent group. In recurring cases, herpes simplex virus DNA reappeared temporarily in the cerebrospinal fluid of two patients. These results show that polymerase chain reaction assay is a useful diagnostic tool for the early and noninvasive diagnosis of herpes simplex encephalitis in children. Results also suggest that a comparatively short duration of acyclovir therapy may be related to a relapse of herpes simplex encephalitis in some children.