Intra-articular therapy with recombinant human GDF5 arrests disease progression and stimulates cartilage repair in the rat medial meniscus transection (MMT) model of osteoarthritis.

OBJECTIVE: Investigation of osteoarthritis (OA) risk alleles suggests that reduced levels of growth and differentiation factor-5 (GDF5) may be a precipitating factor in OA. We hypothesized that intra-articular recombinant human GDF5 (rhGDF5) supplementation to the OA joint may alter disease progression. METHODS: A rat medial meniscus transection (MMT) joint instability OA model was used. Animals received either one intra-articular injection, or two or three bi-weekly intra-articular injections of either 30 µg or 100 µg of rhGDF5 beginning on day 21 post surgery after structural pathology had been established. Nine weeks after MMT surgery, joints were processed for histological analysis following staining with toluidine blue. Control groups received intra-articular vehicle injections, comprising a glycine-buffered trehalose solution. OA changes in the joint were evaluated using histopathological end points that were collected by a pathologist who was blinded to treatment. RESULTS: Intra-articular rhGDF5 supplementation reduced cartilage lesions on the medial tibial plateau in a dose-dependent manner when administered therapeutically to intercept OA disease progression. A single 100 µg rhGDF5 injection on day 21 slowed disease progression at day 63. A similar effect was achieved with two bi-weekly injections of 30 µg. Two bi-weekly injections of 100 µg or three bi-weekly injections of 30 µg stopped progression of cartilage lesions. Importantly, three biweekly injections of 100 µg rhGDF5 stimulated significant cartilage repair. CONCLUSIONS: Intra-articular rhGDF5 supplementation can prevent and even reverse OA disease progression in the rat MMT OA model. Collectively, these results support rhGDF5 supplementation as an intra-articular disease modifying OA therapy.

Peripheral nerve exposure to HIV viral envelope protein gp120 induces neuropathic pain and spinal gliosis.

Painful sensory neuropathy is a common and debilitating consequence of human immunodeficiency virus (HIV). The underlying causes of neuropathic pain are most likely not due to direct infection of the nervous system by active virus. The goal of this study was to determine whether epineural exposure to the HIV-1 envelope protein gp120 could lead to chronic painful peripheral neuropathy. Two doses of gp120 or BSA control were transiently delivered epineurally via oxidized cellulose wrapped around the rat sciatic nerve. Animals were assessed for neuropathic pain behaviors at several intervals from 1-30 days following nerve surgery. Allodynia and hyperalgesia were observed within 1-3 days following gp120 and sustained throughout the testing period. The gp120-exposed sciatic nerve exhibited early but transient pathology, notably axonal swelling and increased tumor necrosis factor alpha (TNF-alpha) within the nerve trunk. In contrast, intense astrocytic and microglial activation was observed in the spinal cord, and this gliosis persisted for at least 30 days following epineural gp120, in parallel with neuropathic pain behaviors. These findings demonstrate that limited peripheral nerve exposure to HIV protein can induce persistent painful sensory neuropathy that may be sustained and magnified by long-term spinal neuropathology.

Alterations in endogenous brain beta-endorphin release by adrenal medullary transplants in the spinal cord.

While transplants of adrenal medullary cells into the spinal subarachnoid space may produce antinociception via inhibition of spinal pain transmission pathways, alterations at higher central nervous system (CNS) centers have not been addressed. Recent findings suggest that prolonged noxious stimulation results in release of endogenous beta-endorphin in the brain, possibly as a compensatory mechanism to reduce nociception. The goal of this study was to determine whether adrenal medullary transplants in the spinal subarachnoid space alter endogenous beta-endorphin secretion in the hypothalamic arcuate nucleus, its principal CNS source. Pain behaviors and arcuate beta-endorphin secretion by microdialysis were monitored during the formalin pain test in animals with spinal adrenal medullary or control transplants. Basal levels of extracellular beta-endorphin were 3-fold higher in adrenal medullary-implanted than in controls. In control animals, formalin induced robust pain behaviors and a marked transient increase in beta-endorphin release 30-60 min following injection. In contrast, pain behaviors were attenuated and the formalin-induced increase in beta-endorphin was completely blocked in adrenal medullary implanted animals. Findings from these studies indicate that adrenal medullary transplants in the spinal subarachnoid space can alter beta-endorphin release in the arcuate nucleus both basally and in response to noxious stimuli. Thus, spinally placed adrenal medullary transplants not only alter local spinal cord pharmacology, but can alter endogenous neurochemistry at higher pain processing centers as well.

Neuropathic pain from an experimental neuritis of the rat sciatic nerve.

Painful peripheral neuropathies involve both axonal damage and an inflammation of the nerve. The role of the latter by itself was investigated by producing an experimental neuritis in the rat. The sciatic nerves were exposed at mid-thigh level and wrapped loosely in hemostatic oxidized cellulose (Oxycel) that on one side was saturated with an inflammatory stimulus, carrageenan (CARRA) or complete Freund's adjuvant (CFA), and on the other side saturated with saline. In other rats, a myositis was created by implanting Oxycel saturated with CFA into a pocket made in the biceps femoris at a position adjacent to where the nerve was treated. Pain-evoked responses from the plantar hind paws were tested before treatment and daily thereafter. Statistically significant heat- and mechano-hyperalgesia, and mechano- and cold-allodynia were present on the side of the inflamed nerve (CARRA or CFA) for 1-5 days after which responses returned to normal. There were no abnormal pain responses on the side of the saline-treated nerve, and none in the rats with the experimental myositis. The abnormal pain responses were inhibited by N-methyl-D-aspartate receptor blockade with MK-801, but were relatively resistant to the dose of morphine tested (10 mg/kg). Light microscopic examination of CARRA-treated nerves, harvested at the time of peak symptom severity, revealed that the treated region was mildly edematous and that there was an obvious endoneurial infiltration of immune cells (granulocytes and lymphocytes). There was either a complete absence of degeneration, or the degeneration of no more than a few tens of axons. Immunocytochemical staining for CD4 and CD8 T-lymphocyte markers revealed that both cell types were present in the epineurial and endoneurial compartments. The endoneurial T-cells appeared to derive from the endoneurial vasculature, rather than from migration across the nerve sheath. We conclude that a focal inflammation of the sciatic nerve produces neuropathic pain sensations in a distant region (the ipsilateral hind paw) and that this is not due to axonal damage. The neuropathic pain is specific to inflammation of the nerve because it was absent in animals with the experimental myositis and in those receiving sham-treatment. These results suggest that an acute episode of neuritis-evoked neuropathic pain may contribute to the genesis of chronically painful peripheral neuropathies, and that a chronic (or chronically recurrent) focal neuritis might produce neuropathic pain in the absence of significant (or clinically detectable) structural damage to the nerve. The model that we describe is likely to be useful in the study of the neuroimmune factors that contribute to painful peripheral neuropathies.

The kappa opioid agonist GR89,696 blocks hyperalgesia and allodynia in rat models of peripheral neuritis and neuropathy.

Previous work demonstrated that, in rats, intrathecal GR89696, a putative kappa-2 opioid receptor agonist, inhibited hyperalgesia to noxious heat in an inflamed hind paw (anti-hyperalgesic effect). Non-inflamed paws were not influenced by kappa-2 receptor activation. The question addressed in this study was whether GR89696 was as effective in blocking hyperalgesia and allodynia in nerve injury models as it was in the inflammation model. GR89696 (6 nmoles, i.t.) completely reversed the hyperalgesia and allodynia observed in both the neuropathy and neuritis models in all sensory tests. However, it did not alter sensory function in non-injured limbs nor in sham operated animals. Naloxone (1 mg/kg, i.p.) reversed the anti-hyperalgesic and anti-allodynic effects of GR89696. The mu agonist DAMGO (6 nmoles, i.t.) and the kappa-1 agonist U69593 (100 nmoles, i.t.) only partially reversed hyperalgesia and allodynia. These findings suggest that kappa-2 opioid receptors may be a useful target for the pharmacological control of hyperalgesia and allodynia.

NMDA-induced spinal hypersensitivity is reduced by naturally derived peptide analog [Ser1]histogranin.

N-methyl-D-aspartate (NMDA) receptor activation is thought to initiate a cellular cascade of events in the spinal cord that leads to neuronal hyperactivation and exaggerated persistent pain behaviors. Previous studies have demonstrated that implantation of adrenal medullary tissue into the spinal subarachnoid space reduces abnormal pain behaviors such as hyperalgesia and allodynia, possibly by intervening in the NMDA hyperexcitability cascade. Histogranin is a 15-amino acid peptide possessing NMDA receptor antagonist activity that has been isolated from adrenal medullary tissue. The present study examined the ability of stable analog [Ser1]histogranin to reduce abnormal pain-related behaviors induced in rats by direct activation of spinal NMDA receptors. The intrathecal injection of NMDA (5.0, 10.0, 20.0 nmol) produced significant thermal and mechanical hyperalgesia and tactile allodynia in a dose-related fashion. [Ser1]histogranin injected intrathecally prior to NMDA injections dose dependently attenuated or completely blocked hyperalgesia and allodynia. In addition, [Ser1]histogranin administration following NMDA-induction of abnormal pain behaviors reversed these effects. These results demonstrate that a naturally derived adrenal medullary neuropeptide can prevent and reverse NMDA-mediated spinal hyperexcitability. The distinct profile and robust activity of [Ser1]histogranin suggest novel alternative approaches in the management of pain and other CNS disorders involving abnormal excitatory neurotransmission.

Nociceptive stimulus induces release of endogenous beta-endorphin in the rat brain.

The hypothesis that the naturally occurring analgesic peptide, beta-endorphin, is released in the brain in response to pain had never been directly validated. In this study, we applied a brain microdialysis method for monitoring beta-endorphin release in vivo, to test this hypothesis in the brains of conscious, freely moving rats. Herein we first show that endogenous beta-endorphin can be measured in vivo in the brain under physiological conditions. Upon induction of a nociceptive stimulus by injection of formalin into the hind-paws of rats, the extracellular levels of beta-endorphin in their arcuate nucleus increased by 88%, corresponding to their nociceptive response. This direct evidence for the release of endogenous beta-endorphin in the brain in response to nociceptive stimulus indicates a possible mechanism for organisms to cope with pain.

Dopamine turnover and metabolism in the striatum of parkinsonian rats grafted with genetically-modified human astrocytes.

The potential of a novel therapeutic approach for treating Parkinson's disease, which involves the transplantation of a transfected human astrocyte cell line SVG-TH, that stably expresses the rate-limiting enzyme for dopamine production, tyrosine hydroxylase, was examined. SVG-TH and untransfected parent cells were grafted into the diseased striatum of rats in which Parkinson's disease had been induced by the administration of 6-hydroxydopamine. The in situ production and spillover of 3,4-dihydroxyphenylalanine (the precursor of dopamine), dopamine and their metabolites in the striatal extracellular fluid of the grafted rats was determined in conscious animals using the microdialysis technique and a high pressure liquid chromatography apparatus. Alleviation of symptoms of Parkinson's disease (abnormal movements) was evaluated by rotation tests. Upon transplantation of the SVG-TH cells into the striatum of the parkinsonian rats, the levels of dopamine in extracellular fluid of the striatum reached those of the normal rats, and correlated well with the improvement (74%) in their rotating behaviour (behavioural deficit). The levels of the two main dopamine metabolites, dihydroxyphenylacetic acid and homovanillic acid, were low in the lesioned rats, even after SVG-TH transplantation. An alternative route of metabolism of dopamine may occur in the transplanted striatum, since the dopamine metabolite, 3-O-methoxy-4-hydroxy-phenylethylamine, appeared, which indicates activity of catechol-O-methyl transferase. Upon blockade of L-aromatic-amino acid decarboxylase, 3,4-dihydroxyphenylalanine accumulated in extracellular fluid of the 6-hydroxydopamine-lesioned and SVG-TH-grafted rats, which indicated that these cells produced active tyrosine hydroxylase in vivo. These findings indicate the potential of treating Parkinson's disease by the intrabrain grafting of human astrocyte cells transfected with the rate limiting enzyme for dopamine production.

NGF involvement in pain induced by chronic constriction injury of the rat sciatic nerve.

Chronic constriction injury (CCI) of the rat sciatic nerve, which within 3 days induces thermal and mechanical hyperalgesia and mechanical allodynia, is used as a model for pain resulting from nerve injury. Involvement of nerve growth factor (NGF) in the development of this hyperalgesia is suggested by the increase in the level of mRNA encoding NGF in cells in the injured area and in dorsal root ganglia at the level of the lesion and the greatly increased NGF levels (determined by ELISA) in the ganglia ipsilateral to the CCI. Application of anti-serum to NGF at the site of CCI delayed the appearance of hyperalgesia, whereas pre-immune serum appeared to enhance it. These results are consistent with the view that NGF is an important factor in the appearance of hyperalgesia associated with unilateral mononeuropathy.

The analgesic effects of R(+)-WIN 55,212-2 mesylate, a high affinity cannabinoid agonist, in a rat model of neuropathic pain.

The effects of a high affinity cannabinoid receptor agonist were evaluated in rats subjected to chronic constriction injury of the sciatic nerve (CCI) or a sham operation. Intraperitoneal (i.p.) injections of the active, but not the inactive enantiomer, alleviated the pain behavior exhibited by CCI animals in a dose dependent manner. Moreover, at doses ranging from 0.43 to 4.3 mg/kg effects on sensitivity to a heat stimulus were observed neither in the paw contralateral to the sciatic ligation, nor in animals subjected to sham surgery. Animals subjected to CCI and treated with 4.3 mg/kg exhibited hypoalgesia in the paw ipsilateral to the ligated sciatic, i.e. heat hypoalgesia was completely reversed. The hypoalgesia is presumed to be the results of unmasking of a sensory deficit reflecting the known loss of C and A delta with CCI. Although side effects were present in some CCI animals subjected to the high dose (4.3 mg/kg), a moderate dose (2.14 mg/kg) completely alleviated the thermal and mechanical hyperalgesia, and mechanical allodynia without side effects. In addition to identifying a potential drug treatment for painful neuropathy, this study suggests that changes in cannabinoid receptors occurs in nerve injured animals.

Increased delayed type hypersensitivity in rats subjected to unilateral mononeuropathy is mediated by neurokinin-1 receptors.

An animal model or peripheral mononeuropathy was utilized in the present study to investigate the potential role of substance P (SP) in modifying immune responses associated with chronic pain conditions. Animals subjected to unilateral sciatic ligation and sham-operated animals were sensitized with keyhole limpet hemocyanin (KLH) and subsequently challenged in the ipsilateral or contralateral hind paw to produce a delayed-type hypersensitivity (DTH) response. Subcutaneous microdialysis and radioimmunoassay were used to measure interstitial fluid SP levels in the challenged tissue prior to and following immune challenge in control and neuropathic animals. Following immune challenge, there was a significant increase in the concentration of SP in tissue dialysate samples from the challenged paw of both sham-operated and neuropathic animals. However, tissue SP levels in neuropathic animals were more than two-fold higher than those obtained from sham-operated controls following challenge. SP concentration remained elevated for 2.5 h following immune challenge in neuropathic animals compared to 90 min in sham-operated animals. Compared with controls, neuropathic animals also exhibited an increased DTH response that was reversed, in a dose-related fashion, by the non-peptide NK-1 receptor blocker L-703,606. The same antagonist had no effect in sham-operated animals. These data suggest that the increased DTH response in animals subjected to unilateral mononeuropathy involves SP and NK-1 receptors present in the challenged tissue.

Spinal cord NMDA receptors modulate peripheral immune responses and spinal cord c-fos expression after immune challenge in rats subjected to unilateral mononeuropathy.

To characterize further the neural involvement in local immune reactions, we evaluated the effect of intrathecal NMDA-receptor blocker dizocilpine maleate (MK-801) on the peripheral immune response itself and on spinal cord c-fos expression induced by the delayed-type hypersensitivity (DTH) response. Immune challenge took place in the hind paw ipsilateral or contralateral to an injured sciatic nerve in both previously sensitized and immune-naive animals. An enhanced immune response was observed bilaterally in the hind paws of animals subjected to unilateral mononeuropathy compared with sham-operated controls. In contrast, no such enhancement was observed when neuropathic animals were challenged in the front paws. The increased DTH response was blocked successfully by the intrathecal administration of an analgesic dose of MK-801. Compared with sham-operated animals, animals subjected to unilateral mononeuropathy showed both a differential distribution and an increase in the number of c-fos-labeled neurons in the dorsal horn of the L3-L5 spinal cord segments after immune challenge. This was observed irrespective of whether the challenge took place ipsilateral or contralateral to the injured nerve. In addition to reversing the changes in immune response, intrathecal administration of MK-801 reversed the pattern of c-fos immunoreactivity in the spinal cord after immune challenge in neuropathic animals. These data suggest that select groups of spinal cord neurons participate in enhancing the peripheral immune response to a specific antigen in neuropathic animals and that this enhancement involves central NMDA receptors.

Electrical stimulation of the sciatic nerve alters neuropeptide content and lymphocyte migration in the subcutaneous tissue of the rat hind paw.

To study the possible mechanism by which peripheral nerves mediate immune responses in target tissues, electrical stimulation of the sciatic nerve was combined with subcutaneous microdialysis of the hind paw. Following unilateral stimulation of the sciatic nerve, an ipsilateral rise in substance P and a bilateral rise in VIP levels were observed in dialysate samples from experimental vs control animals. Electrical stimulation of the sciatic nerve induced a marked hyperemia and swelling of the ipsilateral paw. Quantitative immunocytochemical analysis of paraffin-embedded sections of the hind foot pads demonstrated T lymphocyte migration ipsilateral to the stimulated nerve. These findings suggest that peripheral nerves can directly modulate local immune and inflammatory responses.

Chronic pain and immunity: mononeuropathy alters immune responses in rats.

In order to investigate the possible relationship between chronic pain and the immune system, delayed-type hypersensitivity (DTH) and humoral immunity were assessed in Sprague-Dawley rats subjected to unilateral peripheral mononeuropathy induced by sciatic ligation. Paw withdrawal latency (PWL) time was measured twice during the experiment in animals subjected to sciatic nerve ligation or sham surgery. Sciatic nerve-ligated animals showed hyperalgesia in the leg subjected to neural ligation when compared to the contralateral leg. No differences in PWL times existed in sham-operated animals. In order to exclude possible alterations in immune response due to the surgical procedure or to the hyperalgesia testing, a group of control animals, not subjected to surgical procedures or hyperalgesia testing, was also included in the experiment. Three days post-sciatic ligation or sham surgery, both experimental and control animals were sensitized to keyhole limpet hemocyanin (KLH). A secondary sensitization followed 1 week after the initial immunization. Fourteen days after the initial sensitization, KLH was injected into the hind foot pad and vehicle into the contralateral foot pad in order to assess DTH. One group of rats subjected to sciatic nerve ligation was tested for DTH in the hind foot pad ipsilateral to the ligated nerve, while another group was tested in the contralateral foot pad. Twenty-four hours following foot pad injections, the thickness of both paws was measured and animals were bled to test for anti-KLH immunoglobulins. Animals in which mononeuropathy was induced, but not sham-operated or control animals, exhibited an enhanced DTH response to KLH.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-protective immunity induced by Babesia bovis clones with antigenically unrelated variable merozoite surface antigens.

Babesia bovis merozoite surface antigen 1 (MSA-1) induces antibodies capable of neutralizing merozoites in vitro. Both MSA-1 and the co-expressed MSA-2 are encoded by a polymorphic multigene family and are antigenically variant among strains isolated from widely separated geographic regions. In this study, cross-protective immunity between two B. bovis clones, Mexico Mo-7 and Israel-C, that have antigenically unrelated MSA-1 and MSA-2 surface proteins was assessed. Cattle immunized by infection with either clone were significantly protected against challenge with the uncloned Israel Bbv strain. This indicates that epitopes capable of inducing partial protection are shared among different strains and that immunity is not solely dependent upon MSA-1 or MSA-2. However, cattle immunized with the Israel-C clone, derived from the Israel Bbv strain, were significantly better protected against BbV challenge than were cattle immunized with the Mexico Mo-7 clone bearing antigenically unrelated MSA-1 and MSA-2. The significant difference in immunity induced by the homologous strain versus an antigenically variant strain indicates that epitope variation among strains is relevant to immunity against babesiosis.

Reactivation and aging of phosphorylated brain acetylcholinesterase from fish and rodents.

Species-related differences in sensitivity to acute intoxication by anticholinesterase compounds have been attributed, in large part, to differences in the kinetics of inhibition of acetylcholinesterase (AChE) in vitro. Since inhibition of AchE is also influenced by the stability of the phosphorylated enzyme complex, it was of interest to compare the rates at which the inhibited enzyme from different species subsequently either reactivates or ages. Brain AChE from rats, mice, fathead minnows, or rainbow trout was preincubated with an IC90 concentration of either paraoxon or malaoxon. The first-order rate constants for both the reactivation and aging of paraoxon-inhibited AChE from rats and mice were significantly greater than those observed for either species of fish. Following malaoxon inhibition, however, rodent AChE reactivated more rapidly but aged more slowly than did the enzyme from minnows. Therefore, the data suggest that compared to rodents, intermittent or continuous exposure of fish to sublethal concentrations of anticholinesterase compounds is more likely to result in a cumulative toxicity owing to the relative irreversibility of AChE inhibition.

[Aetiological treatment of benign goitre with l-thyroxine (author's transl)]. / Die kausale Therapie der primär-blanden Struma mit L-Thyroxin.

L-Thyroxine was given to 3956 patients with benign goitre between 1974 and 1977, 2314 of which were controlled over a period of 3 years at regular intervals. Benign goitre in children less than 14 years old and postoperative recurrences were not evaluated. Definite reduction in goitre size wss achieved in 77.2% of patients by continuous long-term treatment over 3 years. In large goitres and those with nodular transformation the status quo was maintained in all but exceptional cases.