NRP-1 Receptor Expression Mismatch in Skin of Subjects with Experimental and Diabetic Small Fiber Neuropathy.

The in vivo cutaneous nerve regeneration model using capsaicin is applied extensively to study the regenerative mechanisms and therapeutic efficacy of disease modifying molecules for small fiber neuropathy (SFN). Since mismatches between functional and morphological nerve fiber recovery are described for this model, we aimed at determining the capability of the capsaicin model to truly mimic the morphological manifestations of SFN in diabetes. As nerve and blood vessel growth and regenerative capacities are defective in diabetes, we focused on studying the key regulator of these processes, the neuropilin-1 (NRP-1)/semaphorin pathway. This led us to the evaluation of NRP-1 receptor expression in epidermis and dermis of subjects presenting experimentally induced small fiber neuropathy, diabetic polyneuropathy and of diabetic subjects without clinical signs of small fiber neuropathy. The NRP-1 receptor was co-stained with CD31 vessel-marker using immunofluorescence and analyzed with Definiens® technology. This study indicates that capsaicin application results in significant loss of epidermal NRP-1 receptor expression, whereas diabetic subjects presenting small fiber neuropathy show full epidermal NRP-1 expression in contrast to the basal expression pattern seen in healthy controls. Capsaicin induced a decrease in dermal non-vascular NRP-1 receptor expression which did not appear in diabetic polyneuropathy. We can conclude that the capsaicin model does not mimic diabetic neuropathy related changes for cutaneous NRP-1 receptor expression. In addition, our data suggest that NRP-1 might play an important role in epidermal nerve fiber loss and/or defective regeneration and that NRP-1 receptor could change the epidermal environment to a nerve fiber repellant bed possibly through Sem3A in diabetes.

Evaluation of pain behavior and bone destruction in two arthritic models in guinea pig and rat.

The primary aim of the study was to describe and correlate pain behavior and changes in bone morphology in animal models of arthritis both in rats and guinea pigs. Either complete Freund's adjuvant (CFA) or mono-iodoacetate (MIA) solution was injected into the left knee joint to obtain a model for rheumatoid arthritis and osteoarthritis, respectively. Subsequently, animals were behaviorally tested during a period of 12 days after CFA injection and at least 19 days after MIA injection. During these observation periods increasing pain behavior was observed, characterized by decreased von Frey mechanical thresholds and weight bearing on the affected limb. In Hargreaves' paw flick test slightly increased thermal hypersensitivity was observed in some instances in guinea pigs. In rats there was also decreased limb-use during forced ambulation. To evaluate bone destruction mu-computed tomography scans of the arthritic knee were taken on the last experimental day. Different bone parameters indicative of osteolysis and decreased trabecular connectivity were significantly correlated with the observed pain behavior. Detailed description of morphological changes in arthritic joints better characterizes the different animal models and might add to the knowledge on the working mechanisms of analgesic compounds that have an influence on bone structures in arthritis.

A preclinical comparison between different opioids: antinociceptive versus adverse effects.

Reduced side-effect liability of opioids may enhance the patient's quality of life and decrease the incidence of opioid-insensitive pain. Literature offers few comparative data between different opioids at equianalgesic doses. Therefore morphine, fentanyl, buprenorphine, codeine, hydrocodone and oxycodone were compared for analgesic properties and side-effect profiles in rats. Analgesic efficacy was analysed using a tail withdrawal test for acute thermal nociception, a formalin test for chemically induced inflammatory pain and a von Frey test for mechanical hypersensitivity. For side-effect profiling inhibition of gastrointestinal activity was evaluated in a charcoal and ricinus oil test, arterial PCO(2) was determined for measuring respiratory depression, the discriminative stimulus properties linked to the narcotic cue were assessed using a drug discrimination learning test, and motor coordination was tested through rotarod performance. ED(50)'s for the occurrence of side-effects were compared to ED(50)'s in behavioural pain tests. Fentanyl had a strong analgesic potency and, compared to other opioids, an acceptable side-effect profiling at analgesic ED(50)'s. Also consistent was the ceiling effect of buprenorphine implying an increased safety margin for side-effects, but a decreased analgesic efficacy. Differences between opioids as observed in this study can have important indications for their use in acute as well as in the onset of chronic treatments.

Bone cancer pain model in mice: evaluation of pain behavior, bone destruction and morphine sensitivity.

The primary aim of the study was to correlate pain development during bone cancer growth with objectively obtained tumor-induced changes in bone morphology. Additionally morphine sensitivity of this bone pain was evaluated. Mice were injected into the femur with osteolytic NCTC2472 cells, and behaviorally followed during a 3-week period. During the observation period increasing pain behavior was observed in tumor-bearing animals. Tumor mice exhibited spontaneous and movement-evoked lifting, the latter evoked through non-noxious palpation of the tumor. Limb use during forced ambulation on a rotarod decreased to substantial non-use of the affected limb by day 23. On day 23, micro-computer tomography scans of the tumor-bearing bones were evaluated for bone destruction. Different bone parameters indicative of osteolysis or fragmentation were significantly correlated with pain behavior. In a separate group of mice the effects of different morphine doses on pain behavior were evaluated on days 17 and 21 of tumor growth. Spontaneous lifting and movement-evoked lifting were sensitive to morphine treatment, although stress-induced analgesia due to repeated restraint might minimize movement-evoked lifting in mice. Limb use during forced ambulation was only slightly ameliorated by high morphine doses.

Morphine-induced analgesia in the hot-plate test: comparison between NMRI(nu/nu) and NMRI mice.

NMRInu/nu mice are frequently used in cancer research, but their use in behavioural pain tests is unexplored. As behaviour of NMRI mice in pain tests is well-documented, a hot-plate test was performed comparing acute thermal nociception in NMRInu/nu and NMRI mice - untreated and morphine-treated - to estimate the usefulness of NMRInu/nu mice for further research on cancer pain. In both strains, morphine dose-dependently increased response latencies, number of animals reaching cut-off times and AUC values. Yet in NMRInu/nu mice, as compared to NMRI mice, all curves were shifted to the right. In order to be comparable, cut-off times must express a similar degree of baseline response augmentations. NMRInu/nu mice had substantially lower pre-drug latencies, indicating a lowered threshold for painful thermal stimuli, therefore effects of morphine in NMRInu/nu mice were also analysed using a lower cut-off time. Doing so, morphine resulted in similar effects in both strains. The effects were independent of hot-plate temperature, because similar results were obtained using temperatures of 50 and 55 degrees. The different morphine sensitivity of NMRInu/nu compared to NMRI mice primarily seems to depend upon differences in thermal threshold, probably induced by the different genotype of both strains. To determine whether cancer alters pain threshold or morphine analgesia, LoVo tumour-bearing NMRInu/nu mice were also tested. The tumour presence had no influence on withdrawal latencies or morphine efficacy. In general it can be concluded that NMRInu/nu mice with or without tumour can be used for nociceptive testing if baseline sensitivity is properly defined.

Immunolocalization of sex steroid hormone receptors in the canine uterine tube and their relation to sex steroid hormone concentrations.

The aim of this immunohistochemical study was to describe the cellular distribution of the estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and androgen receptor (AR) in canine uterine tubes. Samples of uterine tubes were taken from dogs in different stages of the estrous cycle, and dogs that were pregnant or had just delivered. Nuclear staining for sex steroid hormone receptors was observed in the surface epithelium, stromal cells and smooth muscle cells of the muscular layer. Only slight differences in staining pattern were observed between the ampulla and fimbriae. The staining for ERalpha and PR showed changes throughout the estrous cycle. Some of these changes were related to changing concentrations of sex steroid hormones. High staining scores for ERalpha and PR were found during proestrus and low scores during early metestrus. The staining for AR showed only minor cyclic changes. However, during proestrus and estrus, cytoplasmic staining for AR was observed in differentiated secretory epithelial cells, when nuclear staining in these cells was nearly absent. For the three hormone receptors, stromal cells generally stained with a higher intensity than epithelial cells. It is likely that many steroid hormone actions on the epithelium are mediated through stromal cells. During pregnancy, rather high staining scores were found for ERalpha and AR in the uterine tube. This is in contrast to observations in the canine pregnant uterus.

Immunolocalization of sex steroid hormone receptors in canine vaginal and vulvar tissue and their relation to sex steroid hormone concentrations.

The aim of this immunohistochemical study was to describe the cellular distribution of the estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and androgen receptor (AR) in canine vaginal and vulvar tissue. Samples were taken from dogs in different stages of the estrous cycle. Nuclear staining for ERalpha, PR and AR was observed in surface epithelium, stromal and smooth muscle cells. Receptors were also expressed in vulvar skin. Cytoplasmic staining for AR was observed in basal and parabasal cell layers of vulvar and vaginal epithelium. For all three receptors, staining intensity was generally higher in stromal cells compared with epithelial cells, suggesting that stromal cells may be more receptive to steroid hormone action. Therefore, as in other tissues of the female genital tract, stromal-epithelial interactions induced by sex steroid hormones may be of importance in canine vaginal and vulvar tissues. No cyclic changes in receptor immunostaining were observed. Significant positive correlations were found between receptor immunostaining in some vaginal and vulvar cell groups and the serum concentrations of estradiol-17beta and testosterone, but not with the serum progesterone concentration. Significant negative correlations were found between ERalpha immunostaining in epithelial and stromal cells of the vagina and the serum estradiol-17beta concentration, suggesting a negative feedback mechanism between estradiol-17beta and its receptor. Both cell types play a role in the differentiation of vaginal epithelium, under the influence of estradiol-17beta.