A.M. Di Giulio

A mass fragmentographic assay of 3-methoxytyramine in rat brain.

Abstract— A mass fragmentographic method for the measurement of 3‐methoxytyramine, a metabolite of dopamine formed after its neuronal release, is described. This method allows the assay of this dopamine metabolite in the picogram range, thus utilizing only few milligrams of brain areas containing dopaminergic neurons. Moreover, the method is simple and possesses the high specificity intrinsic to mass fragmentography.

Glycosaminoglycans in nerve injury: I. Low doses glycosaminoglycans promote neurite formation

This study has shown that glycosaminoglycans added to the culture medium may affect neurite formation in SH‐SY5Y neuroblastoma cells. The most effective glycosaminoglycans are heparin and COS 8, a preparation with low anticoagulant activity. Promotion of neuritogenesis was remarkable at concentrations as low as 10‐8 and 10‐10. When added at 10‐4 M both agents are inhibitory. Chondroitin‐4 sulfate, dermatan sulfate, and heparan sulfate were also effective, the doses required were, however, as high as 10‐4 M for promoting and 10‐4 M for inhibiting neuritogenesis. Thereby low doses of glycosaminoglycans promote, while higher doses inhibit neurite formation. The effects were observed when neuritogenesis was promoted in neuroblastoma cultures either by deprivation of serum or by addition of retinoic acid, in the former case neuritogenesis occurred within 48 hr; in the latter, in 14 days. PC12 pheochromocytoma cells neuritogenesis was triggered by adding NGF to the culture medium. We have also observed that glycosaminoglycan supplementation to the culture medium lowered the quantity of NGF required to form neurites by PC12 cells. Glycosaminoglycans at the dose of 10‐8 M allow the formation of PC12 neurites even in presence of 1 ng/ml NGF, a dose that normally is ineffective.

Modulation of opioid system in C57 mice after repeated treatment with morphine and naloxone: biochemical and behavioral correlates

C57 BL/6J (C57) mice display a particular pattern of responses following morphine administration, such as a rapid development of tolerance to the pharmacological action of the opiate and an increase in locomotor activity after a single injection of the drug. We have measured met-enkephalin content and the responsiveness of different opiate receptors after repeated administration of morphine and naloxone. Prolonged morphine administration changes neither met-enkephalin levels, nor the density of the opiate receptors in mice brain. In contrast repeated administration of the opiate antagonist naloxone, produced a marked increase in the number of 3H- DHM and 3H- DADLE binding sites in striatum and brainstem without modifying met-enkephalin concentrations. Behavioral studies have indicated that the morphine-induced increase in locomotor activity is enhanced in naloxone pretreated mice, thus suggesting a possible correlation between the behavioral response to morphine in C57 mice and the higher number of opiate receptors in the striatum.

Peripheral nerve lesions cause simultaneous alterations of substance P and enkephalin levels in the spinal cord.

The substance P and Met-enkephalin content were measured in the rat lumbar spinal cord after monolateral section of the sciatic nerve. The proximal stump of the lesioned nerve was either ligated, limiting the formation of neuroma, or sutured intraperitoneally, allowing the formation of a very large neuroma. Both types of lesion caused a similar peptide loss. Substance P and Met-enkephalin decreased by about 50% 10 days following the lesion. Such a loss was maintained even 30 days postoperatively and was not affected by the neuroma size. Immunohistochemical stainings showed that the loss of both peptides occurred in laminae I and II of the dorsal horn. It is suggested that pain sensation developing after peripheral nerve lesion may be due to the intraspinal loss of enkephalin rather than to the neuroma formation.

Opiates and their antagonists modulate luteinizing hormone acting outside the blood brain barrier

The effects of morphine methyl-iodide and naloxone methyl-bromide, two quaternary derivatives of morphine and naloxone, were evaluated on the modulation of luteinizing hormone secretion in intact and gonadectomized rats. Quaternary compounds are effective in modulating LH release, indicating a site outside the blood brain barrier for their action. More precisely, the median eminence and not the pituitary seems to be the site of action of opiates in modulating LH secretion, since the effect of the quaternary derivatives is abolished by surgical ablation of the median eminence.

Fate of autologous dermal stem cells transplanted into the spinal cord after traumatic injury (TSCI)

Rat dermis is a source of cells capable of growing in vitro and, in appropriate conditions, forming floating spheres constituted by nestin-positive cells. We have clonally grown these spheres up to the 15th generation. These spheres can be dissociated into cells that differentiate in vitro under appropriate conditions, these cells are labeled by antibodies to immature neuron markers such as nestin and beta-tubulin III and, later, to mature neuron markers such as microtubule-associated protein 2 and neurofilaments. However, most cells are positive to the astroglial marker glia fibrillary acidic protein (GFAP). When sphere-derived cells are transplanted into the spinal cord after traumatic injury, their migration into the lesion cavity is optimal but their differentiation is dependent upon the time interval between lesioning and cell transplantation. Injection of skin-derived stem cell within 30 min from injury yields mainly membrane activated complex-1 (MAC-1), cluster of differentiation-4 (CD-4) and CD-8 positive cells, that 60-90 days later undergo apoptosis. However, when transplantation is performed 7 days after injury, most cells (65% of total) are positive to staining with antibodies to GFAP, others (16%) to neurofilaments, and a smaller amount (2%) to the endothelial marker, platelet endothelial cell adhesion molecule. Thus our study shows that delayed transplantations of dermis-derived stem cells yield healthy cells that do not die, migrate to the lesion site, and there differentiate mainly in cells expressing glia and neuronal markers. On the other hand there is the possibility of dye transfer from labeled cells to endogenous cells, and this might influence the data.

3-methoxytyramine and different neuroleptics: Dissociation from HVA and DOPAC

Abstract The effect of haloperidol and clozapine, a cataleptogenic and a non cataleptogenic neuroleptic, on striatal concentrations of 3-methoxytyramine (3-MT), DOPAC and HVA has been studied. The data indicate that 3-MT changes seem to parallel the electrophysiological and behavioral profile of these two neuroleptics. Infact, haloperidol and clozapine, given at doses which elicit comparable increase of DOPAC and HVA concentrations, affect differently 3-MT content which is increased by haloperidol but not modified by clozapine. Moreover, the results obtained after various lesions which differently affect the integrity of striatal or nigral neuronal organization, seem to suggest that mechanisms localized in substantia nigra may play an important role in the modulation of the haloperidol induced increase of striatal 3-MT.

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.

Brain β-endorphin concentrations in experimental chronic liver disease

Abstract β-Endorphin, Met-enkephalin, substance P and somatostatin concentrations have been evaluated in brain areas of rats with severe liver disease obtained by chronic pretreatment with CCl 4 for 3 or 9 weeks. β-Endorphin, but not Met-enkephalin, somatostatin or substance P concentrations were significantly decreased in the hypothalamus of both the three and nine week-treatment groups. The β-endorphin decrease we observed might be tentatively attributed to a modification of GABA levels, but not serotonin, since the stimulation of the serotoninergic system induced a significant increase, while the potentiation of the GABAergic system induced a clear decrease of β-endorphin concentrations in the hypothalamus of treated rats.

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.