Delayed treatment with chondroitinase ABC reverses chronic atrophy of rubrospinal neurons following spinal cord injury.

Degradation of extracellular matrix chondroitin sulphate proteoglycans (CSPGs) using Chondroitinase ABC (ChABC) is a promising strategy for the treatment of spinal cord injury, with potent effects on promoting functional recovery and anatomical repair in spinal injured animals. We have previously demonstrated that ChABC treatment prevents atrophy of corticospinal projection neurons following spinal injury in adult YFP-H mice. Here, we investigate whether ChABC-mediated repair of the cell body extends to rubrospinal projection neurons (RSNs), whether neuroprotective effects can be sustained long-term and importantly, whether delayed treatment with ChABC can reverse chronic atrophy. Adult YFP-H mice underwent unilateral rubrospinal tract transection and were treated with ChABC or a control enzyme, delivered either acutely post-injury or after a one month delay. Eight weeks following injury and control treatment, RSNs in the injured red nucleus, identified by YFP label and NeuN immunoreactivity, showed severe atrophy, with ~40% loss of mean cell area compared to uninjured neurons in the contralateral red nucleus. Both acute and delayed treatment with ChABC promoted a significant rescue of injured RSNs, restoring cell area to ~80% and ~70%, respectively, of that in uninjured neurons. Thus, we demonstrate for the first time that CSPG degradation in the injured spinal cord not only promotes sustained rescue of cell atrophy when delivered acutely but can also reverse chronic atrophy in descending projection neurons. Thus, modulation of the extracellular matrix can mediate neuroprotective effects both early and late after spinal cord injury.

Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions.

BACKGROUND: Interruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate. Beside its role in membrane synthesis, oleate could act as a neurotrophic factor, involved in signal transduction pathways via activation of protein kinases C. RESULTS: In situ hybridization and immunohistochemistry demonstrated a strong up-regulation of SCD at mRNA and protein level in regenerating neurons of the rat facial nucleus whereas non-regenerating Clarke's and Red nucleus neurons did not show an induction of this gene. CONCLUSION: This differential expression points to a functionally significant role for the SCD-1 in the process of regeneration.

Induction of microglial reaction and expression of nitric oxide synthase I in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection.

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy.

Regional differences between the immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II alpha and beta isoforms in the brainstem of the rat.

The distribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) alpha and beta isoforms in the brainstem of adult rats was investigated using an immunohistochemical method with two monoclonal antibodies which specifically recognize the alpha and beta isoform, respectively. We found that these isoforms were differentially expressed by neurons in the substantia nigra, red nucleus, dorsal cochlear nucleus, pontine nuclei and inferior olivary nucleus. Neurons in the inferior olivary nucleus express the alpha isoform, but not the beta isoform. In contrast, neurons in the substantia nigra, red nucleus and pontine nuclei were immunostained with the beta antibody, but not with the alpha antibody. In the dorsal cochlear nucleus, neurons in layers I and II were alpha-immunopositive, whereas neurons in layers III and IV were beta-immunopositive. Therefore, the distribution of the CaM kinase II alpha-immunopositive neurons is completely different from that of CaM kinase II beta-immunopositive neurons. Next we examined the possible coexistence of CaM kinase II alpha isoform and glutamate or that of CaM kinase II beta isoform and glutamic acid decarboxylase (GAD) in the single neuron by double immunofluorescence labelling using a pair of anti-alpha and anti-glutamate antibodies, or a pair of anti-beta and anti-GAD antibodies. The results indicated that neurons expressing anti-alpha immunoreactivity were also immunopositive against anti-glutamate antibody, and neurons expressing beta isoform were also immunopositive against anti-GAD antibody, suggesting that alpha-immunopositive neurons are classified as excitatory-type neurons, and on the contrary, beta-immunopositive neurons are classified as inhibitory-type neurons. In conclusion, the present study confirmed that alpha- and beta-isoforms of CaM kinase II are differentially expressed in the nuclei of the brainstem and have different roles.

Expression of choline acetyltransferase immunoreactivity in the rat red nucleus after postnatal enucleation.

The presence of cholinergic neurones in the rat red nucleus (RN) is a controversial subject. We investigated this issue by immunocytochemistry in both adult, normal and neonatally enucleated rats. Choline acetyltransferase (ChAT)-immunoreactive neurones were found in the RN only of enucleated rats. An increase in cytochrome oxidase staining was also observed in the RN of enucleated rats. Our data support the concept that neurones in the rat RN are cholinergic and suggest that their activity could be modulated by the visual input.

Immunohistochemical evidence of plasticity of gamma-aminobutyric acid neurons in the red nucleus and adjacent reticular formation after contralateral cerebellectomy in the adult cat.

A comparative mapping of gamma-aminobutyric acid neurons identified by means of glutamic acid decarboxylase (GAD) immunohistochemistry was performed in the red nucleus (RN) in both intact and hemicerebellectomized adult cats (21 days postoperative). In the deafferented RN (contralateral to the lesion) as well as in the adjacent dorsolateral reticular formation, a marked increase in the number of GAD-positive perikarya was observed. In this mesencephalic area, some neurons may therefore increase their endogenous levels of immunodetectable GAD, as a response to cerebellar deafferentation. This can be viewed as one of the events contributing to functional recovery.

Immunocytochemical demonstration of GABAergic synapses on identified rubrospinal neurons.

GABAergic synapses on rubrospinal neurons were demonstrated with immunocytochemistry combined with intracellular injection of horseradish peroxidase. Sections containing red nucleus neurons were processed for glutamic acid decarboxylase (GAD) immunohistochemistry. GAD-immunoreactive synaptic endings formed synaptic contacts with somata and dendrites of red nucleus neurons and identified rubrospinal neurons. Our observation provides further evidence that GABA acts as an inhibitory transmitter mediating cortically evoked inhibitory postsynaptic potentials in red nucleus neurons.

Increased glutamate decarboxylase activity in the red nucleus of the adult cat after cerebellar lesions.

Glutamate decarboxylase (GAD)activity, a marker for GABAergic structures, was studied in the cat red nucleus. GAD is more concentrated in the rostral than in the caudal third of the structure. GAD levels were measured after chronic unilateral lesions of the cerebellum. Destruction of the dentate area and of the nucleus interpositus induced increases of GAD in the contralateral but not in the ipsilateral red nucleus. Similar changes also occurred in the denervated nucleus ventralis lateralis (VL) and nucleus ventralis anterior (VA) of the thalamus. Results show that loss of the excitatory cerebellar input could lead to changes in inhibitory GABAergic nerve terminals. This increase may be induced transsynaptically within existing neurons or, more likely, additional GAD-containing nerve terminals may be formed by axonal sprouting.

Glutamate decarboxylase distribution in discrete motor nuclei in the cat brain.

The distribution of activity of glutamate decarboxylase (GAD), the enzyme synthesising gamma-aminobutyric acid (GABA), was measured in the cat brain by means of microdissection of the structures from frozen slices and a radioisotopic assay for the enzyme. About 20 cerebral regions were chosen for study because of their role in sensorimotor integration. GAD presented an uneven distribution among these areas. Highest activities were found in the basal ganglia, particularly in the substantia nigra and in the globus pallidus, and to a lesser extent in the cerebellum. Relatively low levels of the enzyme were found in the thalamus and in the cerebral motor cortex. Special detailed studies were made in the caudate nucleus, the substantia nigra, and in the red nucleus for the purpose of defining the intranuclear distribution of their GABAergic innervation. There were only small differences in the rostro-caudal distribution of the enzyme in the head of the caudate nucleus but GAD activity was higher in the ventral than in the dorsal part of the structure. In the substantia nigra, GAD activity was high in both the medial and intermediate thirds of the structure. The GAD activity decreased from the caudal to the rostral part of the nucleus. GAD levels were lower in the caudal part of the red nucleus than in the rostral part. These results indicate that GABA would be present as a putative neurotransmitter in many motor nuclei of the cat brain. In view of the general inhibitory action of this amino acid, this could be related to the presence of inhibitory responses widely distributed in these nuclei as identified by mean of electrophysiological studies. The origin of these GABAergic innervations in many cases remains to be determined.

Acetylcholinesterase activity and acetylcholine effects in the cerebello-rubro-thalamic pathway of the cat.

Unilateral transections of the brachium conjunctivum (BC) of cats resulted, after 2-3 weeks, in marked loss of acetylcholinesterase (AChE) activity from the contralateral red nucleus (RN) and ventral tier nuclei of the thalamus (VA-VL). Significant changes in activity were not observed in other locations. Sensitivity of RN neurons to iontophoretically applied acetylcholine (ACh) was studied under conditions which should maximize ACh sensitivity, including AChE inhibition, but ACh was found to have only a weak depressant effect on excitability or no effect at all. Intravenous physostigmine usually increased spontaneous activity of RN neurons, and sometimes increased potentials evoked by electrical stimulation of cerebellar nuclei, to a small extent. Anticholinergic drugs were found not to influence such evoked responses, except to reverse the effects of physostigmine. It is concluded that ACh is not a major transmitter in the excitatory cerebello-rubral tract in spite of the relationship of AChE to this pathway.

Alterations of the nucleus ruber in 3-acetylpyridine intoxication. A light and electron microscopic study.

3-acetylpyridine (3-AP) given in one single dose near to LD50 causes hydropic-vacuolic degeneration and cellular necrosis of varying degree and extension in the red nucleus of white rats in more than 50 percent of the cases. Less frequently similar alterations have been observed in some neighbouring nuclei of the mesencephalon. The regressive damage is remarkably rare if a smaller dose of 3-AP is given and none if the animal is pretreated with nicotine amide. Ultrastructurally the damage of mitochondria is dominant, they might be transformed to cystic lamellar figures. One reason of the enhanced vulnerability to antimetabolite is the outstanding enzyme activity of the red nucleus. In some of the intoxicated animals the six oxidative enzymes tested histochemically show different type of alterations which probably are related to the regressive damage.

Histochemical studies on the morphology of the Golgi apparatus and on the enzymes of carbohydrate metabolism in various nuclei of the rat mesencephalon.

Detailed histochemical studies have been conducted on the distribution of various enzymes such as thiamine pyrophosphatase, alpha-glucan phosphorylase, hexokinase, glucose-6-phosphate dehydrogenase, aldolase, lactate dehydrogenase and succinate dehydrogenase in various components of the nucleus Edinger-Westphali, nucleus n. oculomotorii, nucleus ruber and nucleus niger of healthy adult male Wistar strain rats. The thiamine pyrophosphatase reaction showed the morphological patterns of the Golgi apparatus characteristic for each nucleus. The Golgi apparatus was well developed in the nucleus Edinger-Westphali, composing a network of highly fenestrated plates in the nucleus n. oculomotorii and nucleus ruber, and a simple network in the nucleus niger. These results indicate that the former three nuclei need a rich energy supply and argue against the possibility that the four nuclei have a secretory role. The neurons of the nucleus Edinger-Westphali may derive their energy mainly from glucose of the circulating blood, but glial cells may serve as energy donators to the neurons in the pars compacta of the nucleus niger, and the neurons of the other nuclei may derive energy from both sources. These conclusions are consistent with the morphological patterns of the Golgi apparatus.

Regional distribution of choline acetyltransferase in the human brain: changes in Huntington's chorea.

A stereotaxic method of tissue sampling has been developed permitting detailed studies of the distribution of choline acetyltransferase (CAT) in brains from controls and from patients suffering from Huntington's chorea. The characteristic pattern of CAT distribution within extra-pyramidal structures is described. In Huntington's chorea, CAT is unevenly reduced in several brain regions particularly in the rostromedial part of the caudate nucleus. The results indicate a preferential degeneration of neostriatal cholinergic neurones in Huntington's chorea.