Letizia Vergani

Systemic administration of insulin‐like growth factor decreases motor neuron cell death and promotes muscle reinnervation

Neonatal sciatic nerve axotomy causes motoneuron death and muscle denervation atrophy. The aim of the present study was to determine whether insulin‐like growth factor‐I (IGF‐I) administration promotes muscle reinnervation and counteracts motor neuron loss after such an injury. Six weeks after sciatic nerve axotomy performed in 2‐day‐old pups, the number of motor neurons, as assessed by retrograde transport of horseradish peroxidase injected into the extensor digitorum longus (EDL) muscle, was reduced from 52 ± 3 to 26 ± 3. Subsequent administration of IGF‐I at the doses of 0.02 mg/kg or 1 mg/kg increased the number of motor neurons to 35 ± 2 and 37 ± 5, respectively. The effect on motoneuron survival was accompanied by improved muscle fibre morphometry and restoration of indirect EDL muscle isometric twitch tension, which was about 80% of control values for both doses of IGF‐I compared with 60% observed with saline treatment. Reinnervated EDL muscle from saline‐treated rats cannot hold tetanic tension, which is, however, achieved after IGF‐I treatment at either dose. Thus, both high and low doses of IGF‐I counteracted motoneuron death and improved muscle reinnervation following neonatal sciatic nerve axotomy. IGF‐I at 5 μg/kg failed to increase muscle reinnervation. J. Neurosci. Res. 54:840–847, 1998.

Effects of low doses of glycosaminoglycans and insulin-like growth factor-I on motor neuron disease in wobbler mouse

In this study we examined the effects of insulin-like growth factor-I (IGF-I) and of glycosaminoglycans (GAGs) on the progressive motor neuron disease in wobbler mice. After clinical diagnosis at age 3 weeks, mice received daily subcutaneous injections of IGF-I, or GAGs, or saline for 3 weeks. The histometric analysis revealed that biceps muscle fiber diameter was reduced in wobbler mice and that treatments with GAGs and IGF-I prevented such a drop. The number of atrophic small fibers was markedly reduced and that of the larger ones augmented. No effects on body growth and biceps muscle weight were observed. The combined AChE-silver staining revealed that both treatments promoted intramuscular axonal sprouting. The typical decline of grip strength in wobbler mice was also prevented. This study suggests that GAGs and IGF-I administrations can retard the onset of motor deficit, and reduce muscle atrophy in wobbler mice.

Glycosaminoglycan Supplementation Promotes Nerve Regeneration and Muscle Reinnervation

This study shows that treatment of rats with exogenous glycosaminoglycans stimulates peripheral nerve regeneration, increases the abundance of mRNAs for myelin proteins and promotes muscle reinnervation. After the sciatic nerve had been crushed the number of regenerating axons in the distal stump was markedly and highly significantly increased by glycosaminoglycan treatment throughout the experimental period. The increased number of axons was correlated with increased axon and fibre (axon + myelin) diameter. The abundance of mRNAs for Po protein and myelin basic protein of regenerating nerves was also affected by treatment with glycosaminoglycans. The increase in mRNA was also observed in the contralateral unlesioned nerve. Such a phenomenon did not occur in saline‐treated rats. Glycosaminoglycan treatment markedly increased the number of muscle fibres reinnervated and accelerated the restoration of muscle twitch tension elicited by nerve stimulation. The effect was particularly evident during the early stages (16 and 21 days after nerve crush) of muscle reinnervation.

GLYCOSAMINOGLYCANS IN NERVE INJURY: II. EFFECTS ON TRANSGANGLIONIC DEGENERATION AND ON THE EXPRESSION OF NEUROTROPHIC FACTORS

Injury to the sciatic nerve leads to the transganglionic degeneration of sensory axons and to the induction of neurotrophins and p75 nerve growth factor receptor synthesis by the denervated Schwann cells. Sciatic nerve axotomy caused a marked loss of substance P and of met‐enkephalin in the lumbar cord. Substance P immunostaining and pre‐proenkephalin mRNA expression were reduced in the dorsal horn layers I and II ipsilaterally to the lesion. Treating rats with low doses (0.25 mg/kg) of heparin or COS 8, a natural glycosaminoglycan mixture with low anticoagulant activity, the peptide loss was prevented and the content increased of about 50% above control values. The effects of COS 8 treatment were also evident on Schwann cells. COS 8 augmented the increase of nerve growth factor, brain‐derived neurotrophic factor, and NT‐3 mRNA expression in the distal stump of the axotomized sciatic nerve. Therefore, it can be concluded that glycosaminoglycans neuroprotective effects on lesioned sensory axons might have been mediated by the dramatic promotion of neurotrophin synthesis. Although the in vitro studies (Lesma et al.: J Neurosci Res, 1996) suggested also a likely direct effect as extracellular matrix components that is not mediated by trophic factors.

Glycosaminoglycans boost insulin-like growth factor-I-promoted neuroprotection : Blockade of motor neuron death in the Wobbler mouse

Wobbler mice display forelimb weakness, altered paw positioning, reduced running speed, muscle atrophy and motor neuron loss; co-treatment with glycosaminoglycans and insulin-like growth factor-I counteracts the progression of the disease. Reportedly, treatment with glycosaminoglycans or insulin-like growth factor-I slows the early stages of progressive forelimb dysfunction in wobbler mice. Our aim was to study whether the combination of these two drugs would result in greater neuroprotective effects. In a group of wobbler mice, combined treatment with daily s.c. administration of 20 microg/kg insulin-like growth factor-I and 1 mg/kg glycosaminoglycans was begun upon diagnosis at three weeks of age and continued for the next six weeks. This treatment halted motor neuron loss and markedly reduced the decay of forelimb muscle morphometry and function. Moreover, the mouse phenotype itself was strikingly improved. The effect of the combination treatment was significantly higher than that of the single drugs, even at a dosage as high as 1 mg/kg insulin-like growth factor-I. The ability of the insulin-like growth factor-I/glycosaminoglycans pharmacological cocktail to arrest the progression of motor neuron disease in wobbler mice and the safety of the low dose of insulin-like growth factor-I used hold promise that this combination might represent a novel approach for the treatment of motor neuron disease and peripheral neuropathies.

Muscle reinnervation following neonatal nerve crush. Interactive effects of glycosaminoglycans and insulin-like growth factor-I

This study shows that glycosaminoglycans promote muscle reinnervation following neonatal sciatic nerve injury. Such an effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy. In addition glycosaminoglycan treatment of lesioned rats increased insulin-like growth factor-I messenger RNA and protein in the reinnervated muscle, and insulin-like growth factor-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of nerve lesioned rats with insulin-like growth factor-I promoted muscle reinnervation and prevention of muscle fibre atrophy, higher levels of insulin-like growth factor-I in the reinnervated muscle and of insulin-like growth factor-I and insulin-like growth factor binding proteins in plasma. These data suggest that glycosaminoglycans are potent stimulants of muscle reinnervation and that their effects may be mediated by increased levels of insulin-like growth factor-I.

Neuroprotection, neuroregeneration, and interaction with insulin-like growth factor-I: Novel non-anticoagulant action of glycosaminoglycans

We present recent developments in the area of glycosaminoglycans (GAGs) and their possible interaction with insulin‐like growth factor‐I (IGF‐I). GAGs are constituents of proteoglycans, and the combination of a core protein and a specific GAG makes a unique proteoglycan with a precise developmental pattern and with the ability to bind growth factors. This process is apparently regulated by the moiety of the peripheral GAGs. The supplementation of GAGs promotes neuritogenesis in vitro and stimulates nerve regrowth and muscle reinnervation, an effect correlated with an increase in trophic factor mRNA expression. In the case of neonatal nerve lesion, there is in addition an enhanced motor neuron survival, accompanied by higher levels of IGF‐I in plasma and denervated muscle. The neurotrophic and neuroregenerative effects of exogenous GAGs were also observed in motor neuron disease in the wobbler mouse. J. Neurosci. Res. 51:559–562, 1998.

Glycosaminoglycans treatment increases IGF-I muscle levels and counteracts motor neuron death: A novel nonanticoagulant action.

The present study shows that sciatic nerve crush in 2‐day‐old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulinlike growth factor‐I (IGF‐I) content in the denervated muscles. EDL muscle denervation‐induced atrophy in saline‐treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 ± 3 to 29 ± 8. In GAGs‐treated 42‐day‐old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 ± 4. Treatment with GAGs selectively affects IGF‐I content in denervated hindlimb muscles, which is augmented from 7.02 ± 0.71 ng/mg tissue to 25.72 ± 0.7 in the EDL and from 3.2 ± 0.18 to a robust 211 ± 9.6 in the soleus. J. Neurosci. Res. 55:496–503, 1999.

Perinatal exposure to morphine: reactive changes in the brain after 6-hydroxydopamine

The effects of neonatal 6-hydroxydopamine treatment on the brain of control rats and of rats perinatally exposed to morphine were examined. Noradrenaline levels were increased in the pons-medulla, mesencephalon and caudate of 8-week-old control rats lesioned with neonatal 6-hydroxydopamine; perinatal morphine treatment prevented such an increase. In the caudate, there was a loss of dopamine and an increase of serotonin following the neurotoxic lesion; exposure to perinatal morphine prevented the serotonin increase. Brain expression of synapsin I mRNA was particularly abundant in cerebral cortex, hippocampus, dentate gyrus and olfactory bulb. In perinatal morphine-treated rats, the expression of synapsin I mRNA was significantly reduced; interestingly, the neonatal treatment with 6-hydroxydopamine normalized its expression. Therefore, brain-reactive neurochemical changes triggered by 6-hydroxydopamine were suppressed by perinatal morphine exposure whereas the association of morphine exposure and 6-hydroxydopamine lesion promoted the normal mRNA expression of the synaptic marker synapsin I.

Exposure to perinatal morphine promotes developmental changes in rat striatum

This study shows that perinatal exposure to morphine promotes developmental changes (up to 8 months of life) in the striatum by up‐regulating concentrations of substance P and met‐enkephalin with changes of prometenkephalin A mRNA expression at the day of birth only. Dopamine metabolism (up to 60 days) is also increased as suggested by the reduced concentrations of dopamine and increased content of 3,4‐dihydroxyphenylacetic acid. Tyrosine hydroxylase mRNA expression is selectively reduced only in the substantia nigra by perinatal morphine. Serotonin content is reduced only during the early postnatal days and is unaffected thereafter. Supplementation of naltrexone to morphine‐exposed rats prevents monoaminergic and neuropeptidergic changes in the striatum, which directly implicates opioid receptors in the developmental changes caused by morphine. The data suggest that perinatal morphine may inhibit met‐enkephalin release, causing accumulation of the peptide without corresponding changes in specific mRNA. Dopamine release may also be increased as indicated by a higher metabolism and consequent reduction of tyrosine hydroxylase mRNA expression in the substantia nigra.