Superior cervical ganglion regenerating axons through peripheral nerve grafts and reversal of behavioral deficits in hemiparkinsonian rats.

The superior cervical ganglion (SCG) has been grafted to the brain of adult rats in an attempt to reverse the parkinsonian syndrome that follows destruction of central dopamine systems. However, the main limitation to this approach is the massive cell death that occurs in the grafted SCG after direct transplantation into the brain. In adult rats, 6-hydroxydopamine (6-OHDA) was stereotactically injected into the right substantia nigra (SN). One month later, dopamine denervation was assessed using the apomorphine-induced rotational test. In rats with a positive test, an autologous peripheral nerve (PN) graft was tunneled from the right cervical region to the ipsilateral parietal cortex. One end of PN graft was sutured to the transected postganglionic branch of the SCG and the other end was inserted into a surgically created cortical cavity. The apomorphine test was repeated at 3 days and again at 1, 3, and 5 months after surgery. The brain, SCG, and PN graft were studied under light and electron microscopy and with the tyrosine hydroxylase immunohistochemical and horseradish peroxidase tracing methods. Three days after grafting, there were no significant differences on the apomorphine test as compared to the preoperative test. Conversely, 1,3, and 5 months after grafting, the number of rotations was reduced by 69% (+/-20.2), 66.6% (+/-17.1), and 72.5% (+/-11.3), respectively. Control rats that received a free PN graft to the brain and underwent section of the postganglionic branch of the SCG did not show significant changes on the apomorphine test after surgery. Histological examination revealed that the PN graft was mostly reinnervated by amyelinic axons of small caliber. Approximately 40% of the SCG neuronal population that normally projects to the postganglionic branch survived axotomy and regenerated the transected axons into the PN graft. Axons arising from the SCG elongated the whole length of the graft, crossed the graft-brain interface and extended into brain regions adjacent to the denervated striatum up to 2037 micrometer from the graft insertion site. This work shows that the ingrowth of catecholamine-regenerating axons from the SCG to dopamine-depleted brain parenchyma significantly reduces behavioral abnormalities in hemiparkinsonian rats. This effect cannot be ascribed either to the brain cavitation or to the PN tissue placement in the brain.

Experimental repair of the oculomotor nerve: the anatomical paradigms of functional regeneration.

In adult guinea pigs, the oculomotor nerve was sectioned proximally (at the tentorial edge) or more distally (at the orbital fissure) and immediately repaired by reapproximation. During a 24-week postoperative period, extrinsic eye motility was assessed by analyzing the vestibulo-ocular reflexes. The regenerated oculomotor nerve was studied morphometrically on semi-thin histological sections at 16 and 24 weeks postinjury. The selectivity of muscle reinnervation was investigated by injection of both single (horseradish peroxidase) and double (fluorescent dyes) retrograde axonal tracers into the eye muscles. Following proximal repair of the oculomotor nerve, the degree of recovery of extraocular motility varied among different animals and remained stable over long-term observations. In animals with poor recovery, aberrant eye movements were always found, and the somatotopic map of the reinnervated eye muscles was greatly altered. Distortions of the central representation were also seen in those animals in which a good level of functional recovery was seen. However, in animals with good recovery, a topographic bias was re-established by about 65% of the original neuronal population, as opposed to 26% in the animals with poor recovery. Neurons located contralateral to the axotomized nucleus sprouted intra-axially and projected their axons to denervated eye muscles. The number and diameter of the regenerated axons, the number and soma diameter of the axotomized neurons, and the ratio of distal axonal branches to proximal supporting neurons were all related to the degree of functional recovery. Following repair of the oculomotor nerve at the orbital fissure, extraocular motility had recovered in all of the animals at 16 weeks without aberrant phenomena. Functional regeneration of the distally transected oculomotor nerve is thought to be the result of selective muscle reinnervation.

Levocarnitine acetyl prevents cell death following long-term section of the vagus nerve in rats.

Levocarnitine acetyl has previously been found to significantly prevent axotomy-induced cell death in the spinal cord motor nucleus 9 and 12 months after section of the sciatic nerve in rats. In the present paper, the effects of levocarnitine acetyl on axotomy-induced cell death in the brain stem motor nuclei 90 days after section of the vagus nerve were studied. The right vagus nerve was cut at the neck. To prevent regeneration, a 5 mm-long segment of the vagus nerve was excised and the distal stump was displaced caudally. After surgery, a group of rats (n = 6) was treated with levocarnitine acetyl dissolved in the drinking water (75 mg/kg/day) (Group I). A second group of operated rats (n = 4) received drinking water alone. (Group II). Ninety days postoperatively, in the rats of both groups the proximal nerve stump of the vagus nerve was injected with horseradish peroxidase to label retrogradely the brain stem motoneurons of the dorsal motor vagal and the ambiguus nuclei. The brain stem nuclei were also labelled by horseradish peroxidase in three unoperated control rats (Group III). In the Group II rats, the number of horseradish peroxidase-labelled motoneurons of the dorsal motor vagal nucleus was found to be significantly smaller than in either the Group I (p < 0.01) or the Group III (p < 0.02) animals. In the Group I rats, the number of motoneurons of the dorsal motor vagal nucleus was not significantly smaller compared to the Group III rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the degenerative and regenerative phenomena occurring after transection and repair of the sciatic nerve in rats: effects of acetyl-L-carnitine.

The effects of acetyl-L-carnitine on some degenerative and regenerative phenomena following sciatic nerve transection in rats, were studied. In Experiment 1, acetyl-L-carnitine was administered intraperitoneally at the dose of 50 mg/kg/day for 28 and 56 days following transection and microsurgical repair of the sciatic nerve. On day 56, the acetyl-L-carnitine-treated rats showed a significantly (p less than 0.05) better motor recovery ("clinical assessment") of the peroneal component of the sciatic nerve than the control rats. Twenty-eight days after nerve repair, the acetyl-L-carnitine-treated rats showed a significantly higher (p less than 0.05) number of myelinated axons in the postlesional nerve stump than control rats. Finally, the treated rats had a significantly lower (p less than 0.05) presence of atrophic fibres in the extensor digitorum longus muscle. In Experiment 2 the sciatic nerve was cut. To prevent spontaneous regeneration, a metallic clip was applied to the distal nerve stump and then the nerve stumps were positioned in different anatomical compartments. After surgery, a group of rats was treated with acetyl-L-carnitine dissolved in the drinking water (75 mg/kg/day). Another group of rats received normal water and served as the control group. Three, 6, 9, 12 and 18 months postoperatively, in the rats of both groups, the proximal sciatic nerve stump was injected with horseradish peroxidase to label the spinal cord neurons of the sciatic nerve nucleus. While in untreated rats the number of horseradish peroxidase-labelled neurons decreased with the increase in denervation time, in acetyl-L-carnitine-treated rats the number of horseradish peroxidase-labelled neurons remained stable for as long as 12 months of denervation and decreased only after 18 months of denervation. Furthermore, acetyl-L-carnitine-treated rats showed a significantly higher (p less than 0.05) number of horseradish peroxidase-labelled neurons with respect to untreated rats both after 9 and 12 months of denervation. In Experiment 3, the sciatic nerve was cut and then repaired after periods of 3, 6, 9, 12, and 18 months. Four months after nerve repair, the sciatic nerve was again cut and the proximal nerve stump was injected with horseradish peroxidase to label the spinal cord neurons of the sciatic nerve nucleus. Both acetyl-L-carnitine-treated and untreated rats showed a tendency to have an increased number of horseradish peroxidase-labelled neurons with respect to intact rats of correspondent ages.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on embryonic transplants to the transected spinal cord of adult rats.

Spinal cord tissue was obtained from 13- and 14-day embryonic rats and homologously grafted to the completely transected spinal cord of adult rats. Eight and 12 weeks after grafting, clinical, electrophysiological, histological, and neuroanatomical studies were performed. Motor performance of the hosts was assessed by the inclined-plane test. The conduction of nerve impulses across the lesion-transplantation site was evaluated by recording the spinal corticomotor and somatosensory evoked potentials. The survival, growth, differentiation, and parenchymal integration of the graft were documented histologically on semi-thin sections. The axonal interactions between the host spinal cord and the graft as well as the posttraumatic retrograde degeneration of corticospinal axons were investigated using the horseradish peroxidase (HRP) technique. Clinical and electrophysiological assessments did not demonstrate any functional activity of the graft. On histological examination, grafted neurons showed a survival rate of 55%. Such neurons exhibited a limited degree of growth and differentiation. The extent of parenchymal integration between the host spinal cord and the graft varied considerably among different specimens and in the various regions of every specimen. The HRP investigations demonstrated that some axonal interactions between the host spinal cord and the graft had occurred. Regenerated axons arising from both the spinal cord and the dorsal root ganglia of the host entered the graft and elongated in it. Also, axons from the grafted neurons were able to grow for some distance in the host spinal cord. The phenomenon of the posttraumatic retrograde degeneration of corticospinal axons was not affected by this embryonic tissue grafting.

The third nerve transection and regeneration in rats with preliminary results on the sixth nerve transection and regeneration in guinea pigs.

The relationship between the phenomenon of the nonselective reinnervation and the functional recovery after section and repair of the highly organized third cranial nerve motor system in rats was studied. The same relationship after section and repair of the more simply organized sixth cranial nerve motor system in guinea pigs is presented as preliminary results. Anatomical demonstration of nonselective reinnervation was obtained by injecting horseradish peroxidase (HRP) into the extraocular muscles. A bilateral reinnervation of previously ipsilateral innervated muscles both in the third and the sixth nerve was interpreted as a plastic response of the brain stem neurons to the nerve injury. Functional recovery, evaluated by measuring with an infrared light technique the horizontal and vertical vestibulo-ocular reflexes, was excellent for the rectus lateralis muscle while it was relatively poor i.e. partial for the muscles depending on the third nerve. These data suggest that one of the most important factors influencing the functional recovery after section and repair of a peripheral nerve is the complexity of the nerve motor system organization.

[Cholinergic enzymes in the ciliary ganglia of the chicken and pigeon]. / Enzimi colinergici nel ganglio ciliare di pollo e di piccione.

In the present paper we have comparatively analyzed acetylcholinesterase (AChE) and cholinacetyltransferase (ChAT) activity in chick and pigeon ciliary ganglion. AChE specific activity in the pigeon ciliary ganglion is remarkably higher than the one occurring in the chick; conversely the ChAT specific activity is similar in the chick as well as in the pigeon. Higher AChE activity found in the pigeon ciliary ganglion can be partially attributed to a selective accumulation of the enzyme in already described membrane-limited formations typical of the choroid neurons. After post-ganglionic axotomy such formations undergo a progressive disappearance which parallels the decrease of AChE activity. The present data suggest the hypothesis that the structures under investigation as well as ganglionic AChE are possibly controlled through a retrograde mechanism by their target organ.

[Location of acetylcholinesterase in axotomized ciliary ganglia]. / Localizzazione dell'acetilcolinesterasi nel ganglio ciliare assotomizzato.

In the present note we have investigated the cytochemical localization of acetylcholinesterase (AChE) in the chick ciliary ganglion (CG) after post-ganglionic axotomy obtained by ablation of the eyeball. Preliminary results show at quite early stages after axotomy a remarkable reduction of cytoplasmic AChE, the residual one being localized in the rough endoplasmic reticulum. On the contrary synaptic areas, in particular those concerning the calyciform synapses, still show a marked AchE activity, similarly to what observed in physiological conditions. The decrease of cytoplasmic AChe in axotomized CG does suggest the possibility that such AChE undergoes to a topographical rearrangement moving towards the synaptic areas of ganglionic neurons.

Acetylcholinesterase localization at synapses in chick embryo ciliary ganglion.

The cytochemical localization of acetylcholinesterase (AChE) at calyciform synapses of the chick ciliary ganglion during embryonic development has been investigated. AChE activity is present at the surface membrane of newly formed calyciform synapses and closely follows the progressive enlargement of the synaptic area. The occurrence of a retrograde iris-dependent influence on ganglionic AChE is considered. AChE seems to be a suitable marker for synaptic maturation.

Developmental distributive pattern of acetylcholinesterase in chick embryo ciliary ganglion.

AChE cytochemistry was performed in the chick ciliary ganglion (CG) during various embryonic stages. AChE first appeared in the RER of the neurons at 5 days of incubation (d.i.). Synaptic AChE appeared only later, parallely to the appearance of the calyciform synapses, i.e. at 9 d.i. At first AChE was mainly localized at the calyx side facing the satellite cell, thereafter extending to the neuronal side and especially labeling synaptic contacts occurring at points along the presynaptic membrane. Finally, at 15 d.i., i.e. when the calyx is morphologically mature, AChE reaction labels the whole contour of the calyciform nerve terminal. At 10 d.i., limited AChE-positive extrasynaptic areas of the ciliary neurons surface first appeared, thereafter extending up to affect at 15 d.i. large neuronal surfaces. Some hypotheses can be drawn from our results: (i) the earliest appearance of cytoplasmic AChE seems somehow independent of the establishing of functional synaptic contacts; (ii) the pattern of development of neuronal AChE suggests the existence of a sort of transynaptic control by presynaptic nerve terminals. However, it is possible that concomitant retrograde iris-dependent influences on ganglionic AChE concur in modulating neuronal AChE; (iii) the functional role possibly played by AChE localized at extra-synaptic level still remains to be clarified.

Surface AChE in the chick ciliary ganglion neurons: Ultrastructural localization and possible relations to ?-bungarotoxin receptors.

The localization of acetylcholinesterase activity in the chick ciliary ganglion was investigated by ultrastructural cytochemistry. Both ganglionic cell populations, i.e. the ciliary and the choroid neurons, showed similar distribution patterns of the enzymic activity in the cytoplasm as well as at the neuronal surface. As indicated by specific inhibition tests, the whole enzymic activity was attributable to specific acetylcholinesterase. While the endocellular activity was mainly localized in the rough endoplasmic reticulum, the surface activity occurred at postsynaptic level and at extrasynaptic areas, where the neuronal membrane comes into contact with the plasma membrane of the satellite cell (boundary neuron-satellite cell). Enzymic activity also uniformly occurred at the surface of preganglionic nerve terminals. The surface localization of specific acetylcholinesterase recalls that recently described for ?-bungarotoxin receptors, which suggests that acetylcholinesterase and ?-bungarotoxin receptors can be distributed together, not only at postsynaptic level but also in extrasynaptic neuronal areas and at presynaptic level. The possibility that ?-bungarotoxin receptors and acetylcholinesterase form a .receptive' system not engaged in ganglionic transmission and not exclusively confined to postsynaptic level is discussed in relation to the electrophysiological data existing in literature.

[Histochemical localization of acetylcholinesterase in the chick ciliary ganglion during development]. / Localizzazione istochimica dell'acetilcolinesterasi nel ganglio ciliare di pollo durante lo sviluppo.

The ultrastructural localization of AChE activity has been investigated in the chick ciliary ganglion during embryonic development. Such activity first appears in a relatively early embryonic stage, i.e. at the 6th day of incubation, in the differentiating cisternae of the rough endoplasmic reticulum. In the period between 10 and 15 days of incubation, the reaction product progressively increases extending to other neuronal areas as Golgi complexes, the perinuclear cisterna and some rare areas of the plasma membrane. In particular a certain amount of AChE appears to occur at the neuronal surface where it is contacted by calyciform synaptic nerve endings. This is conceivably to correlate with the morpho-functional maturation of ganglionic synapses.

[Acetylcholine receptors and acetylcholinesterase activity in cultured chick ciliary ganglia]. / Recettori per l'acetilcolina e attivita' acetilcolinesterasica nel ganglio ciliare di pollo in coltura.

Acetylcholine receptor number and the acetylcholinesterase activity have been studied in organ-cultured chick ciliary ganglia. Receptor number decreases progressively reaching minimum value after two days of culture. Acetylcholinesterase activity decreases slowly reaching at day 3 the 75% of the original value. The biological significance of these data is discussed in relation to the ganglion disconnection from the periphery and from the central nervous system.

[Preliminary observations on ciliary ganglia in organ culture]. / Osservazioni preliminari sul ganglio ciliare in coltura d'organo.

Preliminary observations have been made concerning some ultrastructural aspects of the choroid neurons in organ-cultured ciliary ganglia. Reactive nuclear as well as cytoplasmic modifications have been described. They consist mainly of nuclear dense bodies, arrays of smooth endoplasmic reticulum cisternae, clusters of condensed mitochondria. These modifications would confirm the existence of an interaction occurring between the neurons of the ciliary ganglion and their target organs.

[Ultrastructural observations on choroid neurons of ciliary ganglia]. / Osservazioni ultrastrutturali sui neuroni coroidei del ganglio ciliare.

In the adult pigeon ciliary ganglia peculiar membrane-limited structures were constantly observed in the peripheral cytoplasm of choroid neurons. They are continuous with profiles of the endoplasmic reticulum and/or interconnected with each other. In addition they contain a marked acetylcholinesterase histochemical reaction. These data on the whole suggest that these structures are an integral part of the endoplasmic reticulum system, possibly acting as storing sites of proteic material moving along the pathways of the endoplasmic reticulum system.