Rescue and regeneration of injured peripheral nerve axons by intrathecal insulin.

Insulin peptide, acting through tyrosine kinase receptor pathways, contributes to nerve development or repair. In this work, we examined the direction, impact and repertoire of insulin signaling in vivo during peripheral nerve regeneration in rats. First, we demonstrated that insulin receptor is expressed on lumbar dorsal root ganglia neuronal perikarya using immunohistochemistry. Immunoblots and polymerase chain reactions confirmed the presence of both alpha and beta insulin receptor subunits in dorsal root ganglia. In vivo and in vitro assessment of dorsal root ganglion neurons showed preferential localization of insulin receptor to perikaryal sites. In vivo, intrathecal delivery of fluorescein isothiocyanate-labeled insulin identified localization around dorsal root ganglia neurons. The direction and impact of potential insulin signaling was evaluated by concurrently delivering insulin or carrier over a 2 week period using mini-osmotic pumps, either intrathecally, near nerve, or with both deliveries, following a selective sural nerve crush injury. Only intrathecal insulin increased the number and maturity of regenerating sensory sural nerve axons distal to the crush site. As well, only intrathecal insulin rescued retrograde loss of sural axons after crush. In a separate experiment, insulin also rescued retrograde loss and atrophy of deep peroneal, largely motor, axons post-injury. Intrathecal insulin increased the expression of calcitonin-gene-related peptide in regenerating sprouts, increased the number of visualized regenerating fiber clusters, and reduced downregulation of calcitonin-gene-related peptide in dorsal root ganglia neurons. Insulin delivered intrathecally does not appear to influence expression of insulin-like growth factor-1 at dorsal root ganglion neurons or near peripheral nerve injury, but was associated with upregulation of insulin receptor alpha subunit in dorsal root ganglia. Intrathecal insulin delivery was associated with greater recovery of thermal sensation and longer distances to stimulus response with the pinch test following sural nerve crush. Insulin signaling at neuron perikarya can drive distal sensory axon regrowth, rescue retrograde alterations of axons and alter axon peptide expression. Moreover, such actions are associated with upregulation of its own receptor.

Remote neurotrophic support of epidermal nerve fibres in experimental diabetes.

AIMS/HYPOTHESIS: The support of distal regenerating axons and epidermal nerve fibres through growth factor delivery may depend on the site of delivery. While low-dose systemic insulin provides trophic support for regenerating axons or axons from diabetic animals, its potential action upon the most distal neurites within the epidermis is unknown. In diabetic neuropathy, distal loss of axons is an important clinical and pathological feature. We hypothesised that insulin and IGF-1 delivered intrathecally could support the most distal epidermal nerve fibres. MATERIALS AND METHODS: As insulin and IGF-1 receptors are present upon sensory ganglion perikarya, we studied the impact of intrathecal delivery of low-dose insulin and equimolar IGF-1 on the density of epidermal axons expressing protein gene product 9.5 in experimental diabetic rats. After 2 months of diabetes induced by streptozotocin injection, intrathecal delivery of low-dose insulin or IGF-1 or saline was provided for 1 month, with comparison to compatible doses of subcutaneous insulin delivery. RESULTS: Diabetes, in itself, was associated with a decline in epidermal nerve fibre density. Delivery of both intrathecal IGF-1 and insulin was associated with significant improvement in epidermal fibre density (greatest with IGF-1) and length relative to placebo. CONCLUSIONS/INTERPRETATION: Central intrathecal delivery of IGF-1 and insulin offers remote support for epidermal nerve fibres, subjected to 'dying-back' in early diabetic polyneuropathy.

Diabetes mellitus and the sensory neuron.

Sensory neurons in diabetes may be primarily targeted by diabetes and their involvement may account for prominent sensory loss and pain in diabetic patients. Previous studies demonstrating evidence of excessive polyol flux, microangiopathy, and oxidative stress involving sensory axons and ganglia have been joined by more recent work demonstrating altered neuron phenotype, mitochondrial dysfunction, ion channel alterations, and abnormal growth factor signaling. As such, an interesting and unique panoply of molecular changes in primary sensory neurons has been identified in diabetic models. Insulin deficiency and subsequent changes in second messenger signaling may also play an important role in how sensory neurons respond to diabetes. Applying approaches to support sensory neurons in diabetes may be an important therapeutic direction in diabetic patients.

Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway.

Using a combined pharmacological and gene-deletion approach, we have delineated a novel mechanism of neurokinin-1 (NK-1) receptor-dependent hyperalgesia induced by proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor expressed on nociceptive primary afferent neurons. Injections into the paw of sub-inflammatory doses of PAR2 agonists in rats and mice induced a prolonged thermal and mechanical hyperalgesia and elevated spinal Fos protein expression. This hyperalgesia was markedly diminished or absent in mice lacking the NK-1 receptor, preprotachykinin-A or PAR2 genes, or in rats treated with a centrally acting cyclooxygenase inhibitor or treated by spinal cord injection of NK-1 antagonists. Here we identify a previously unrecognized nociceptive pathway with important therapeutic implications, and our results point to a direct role for proteinases and their receptors in pain transmission.

Systemic production of human granulocyte colony-stimulating factor in nonhuman primates by transplantation of genetically modified myoblasts.

Clinical use of human granulocyte-colony stimulating factor (hG-CSF) to treat various diseases involving neutropenia has been previously shown to (1) successfully increase circulating neutrophils, (2) reduce condition-related infections, and (3) cause few side effects in patients. To alleviate the symptoms of neutropenia, the patient must receive frequent injections of recombinant hG-CSF. Permanent ways to deliver stable levels of the molecule to the patient are being investigated. Among them, the transplantation of hG-CSF-secreting cells has been proposed and performed successfully in rodents, using fibroblast cell lines and primary muscle cells. We thus investigated whether similar results could be obtained by intramuscular myoblast transplantation in a large animal model. When 1-3 x 10(8) myoblasts were injected into three Macaca mulatta, hG-CSF was detected at high levels (300-900 pg/ml), which in turn led to a four- to fivefold increase in circulating neutrophils. However, both the concentrations of hG-CSF and neutrophil levels were found to decrease over time. Nonetheless, neutrophils were found at higher levels from the fourth week until the end the experiment (up to 29 weeks) in G-CSF monkeys compared with control animals. These results show that transplantation of hG-CSF-secreting myoblasts may indeed be a therapeutic option for the treatment of neutropenic patients.

Successful myoblast transplantation in fibrotic muscles: no increased impairment by the connective tissue.

BACKGROUND: Implantation of normal myoblasts may eventually be a treatment for inherited myopathies such as Duchenne muscular dystrophy. METHODS: We report a comparative study of the effectiveness on myoblast implantation: (1) into the muscles of young (2 months) mdx mice nonirradiated and noninjected with notexin (group 1), (2) into muscles of old mdx mice (15 months) nonirradiated and noninjected with notexin (group 2), and (3) into muscles of 5 months mdx mice irradiated 3 months before the transplantation (group 3). Roughly 3 million cells were injected with bFGF in the Tibialis anterior. RESULTS: Although mice of groups 2 and 3 had significantly more (P<0.05) fibrotic tissue in their muscles than those of group 1, the transplantation success was not significantly different among the three groups. CONCLUSION: Therefore these results demonstrated that myoblast transplantation can be successful even when there is abundant fibrosis.

Normal myoblast implantation in MDX mice prevents muscle damage by exercise.

One consequence of the lack of dystrophin is a higher vulnerability of myofibers to eccentric exercise. In this study, we compared the effect of downhill running on Biceps brachii of MDX mice with or without transplantation of normal myoblasts. Exercise induced damaged was detected by Evans blue staining. In control MDX mice, 26.3% of the fibers were permeated by this dye, myoblast transplantation prevented such necrosis. In the transplanted muscles, only dystrophin negative fibers were injured. Indeed, in muscles containing at least 40% dystrophin positive fibers, the damage was significantly reduced in the grafted muscle. Thus the transplantation of normal myoblasts increases the resistance of dystrophic muscles to exercise. Our results suggest that transplantation of normal myoblasts to DMD patients may have beneficial effects.

Evidence of mdx mouse skeletal muscle fragility in vivo by eccentric running exercise.

Duchenne muscular dystrophy is an X-linked devastating disease due to the lack of expression of a functional dystrophin. Unfortunately, the dystrophin-deficient mdx mouse model does not present clinical signs of dystrophy before the age of 18 months, and the role of dystrophin in fiber integrity is not fully understood. The fragility of the skeletal muscle fibers was investigated in transgenic mice expressing beta-galactosidase under the control of a muscle specific promoter. Adult mdx/beta-galactosidase (dystrophin-negative) and normal/beta-galactosidase (dystrophin-positive) mice were submitted to one short session of eccentric, downhill running exercise. The leakage of muscle enzymes creatine kinase and beta-galactosidase was investigated before, 1 h after, and 3 days after the running session. A significant and transient rise in the level of these enzymes was noted in the serum of mdx mice following the exercise session. Thus, the lack of dystrophin in the mdx model led to local microdamages to the exercised muscle allowing leakage of proteins from the fibers. The peak leakage was transient, suggesting that muscle fiber lesions were rapidly repaired following this short, noninvasive eccentric running session.

Muscle fibers of mdx mice are more vulnerable to exercise than those of normal mice.

It is well known that eccentric exercise induces muscle damage by disrupting the sarcolemma. The aim of this study was to analyze the effects of downhill running on several locomotor and respiratory muscles of normal and mdx mice. Degenerating muscle fibers in the skeletal muscles of mice were visualized by in vivo staining with Evans blue. This dye injected intravenously stained only degenerating muscle fibers which were visible as blue fibers macroscopically and could also be seen as red fluorescent fibers microscopically. Evans blue-stained muscle fibers were either hypercontracted or degenerating. Without exercise no muscle fibers were labeled with Evans blue in the normal mice, indicating that their membranes were intact. However, even without exercise, the percentage of fibers permeable to Evans blue varied from 2% to 15% in various muscles of the mdx mice. Our downhill running protocol (i.e., running down a treadmill with a 15 degrees slope at 10 m/min) produced in normal mice only a slight (0-3%) increase in percentage of muscle fibers which were permeable to the dye compared with up to 31% in some mdx muscles.

[Dystrophin expression in skeletal muscles in endurance-trained hypo-hyperthyroid and growth-hormone treated rats]. / Expression de la dystrophine dans le muscle squelettique du rat entraîné en endurance, hypo-hyperthyroïdien, et traité par l'hormone de croissance.

The aim of this study was to determine the effect of chronic muscular exercise, thyroid status and growth hormone administration on skeletal muscle dystrophin expression. Relative abundance of dystrophin (quantity in arbitrary units/50 micrograms of protein) was measured by immunoblotting and densimometry. Our results indicate that relative abundance of dystrophin in slow-or fast-twicht muscle was not modified by chronic muscular exercise (5 weeks), thyroxine administration (4 weeks), antithyroid drug treatment (6 weeks) or growth hormone administration (6 weeks).