Antal Nógrádi

The effect of riluzole treatment in rats on the survival of injured adult and grafted embryonic motoneurons

The effect of riluzole on the survival of injured motoneurons was studied. The L4 ventral root was avulsed and reimplanted into the spinal cord. Immediately after the operation, 4 animals were treated with riluzole for 3 weeks while another 4 animals received no treatment after the operation. Three months later the fluorescent dyes, Fast Blue and Diamidino Yellow, were applied to the cut ventral ramus of the L4 spinal nerve, for retrograde labelling of neurons. Three days later, the spinal cords were processed to reveal the retrograde‐labelled cells. In untreated animals, there were 20 ± 2.1 labelled neurons (± SEM), while in animals treated with riluzole there were 723 ± 26. Thus, treatment with riluzole dramatically enhanced the survival of injured motoneurons. In another series of experiments, after avulsion of the L4 ventral root and its reinsertion, embryonic spinal cord pieces were grafted into the host cord. Five animals received riluzole treatment and 4 were left untreated. In the untreated animals, 125 ± 5.1 retrograde‐labelled cells of both graft and host origin were detected. In rats treated with riluzole, 645 ± 35.7 retrograde‐labelled cells were seen and almost all of these were of host origin. Thus, treatment with riluzole enhanced the survival of injured host motoneurons, and by doing so, (i) reduced the ability of grafted neurons to extend their axons into the reimplanted L4 ventral root, and (ii) reduced the survival of the grafted cells.

Localization of acetazolamide-resistant carbonic anhydrase III in human and rat choroid plexus by immunocytochemistry and in situ hybridisation

Carbonic anhydrase is an essential metabolic enzyme of the central nervous system and has an important role in the production and regulation of cerebrospinal fluid. Although it has been known for over 30 years that inhibition of the enzyme with acetazolamide dramatically but not completely reduces the production of cerebrospinal fluid, the precise mechanism of the inhibitory action has been only recently revealed. In this study we present evidence that apart from carbonic anhydrase II, the catalytically highly active isozyme, carbonic anhydrase III, an acetazolamide-resistant and kinetically different isozyme could be demonstrated in the epithelial cells of the developing and mature rodent and human choroid plexuses. Both isozymes express intense immunostaining revealed with specific antisera, and by using in situ hybridisation histochemistry, carbonic anhydrase III mRNA was also observed. Since the kinetic properties and proportion of brain carbonic anhydrase III in the human choroid plexus are not revealed the function of this isozyme in choroid plexus is still to be determined.

Improved Motor Function of Denervated Rat Hindlimb Muscles Induced by Embryonic Spinal Cord Grafts

Loss of motoneurons results in a decrease in force production by skeletal muscles and paralysis. Although it has been shown that missing motoneurons of rats can be replaced by embryonic homotopic neurons, attempts to guide their axons to their target muscles that have lost their innervation have been unsuccessful. In this study attempts were made to guide axons from grafted embryonic motoneurons to their target via a reimplanted ventral root. Adult hosts that received an embryonic graft prelabelled with 5‐bromo‐2′‐deoxyuridine had their L4 ventral root avulsed and reimplanted into the spinal cord. Three to six months later, neurons that had their axons in the L4 ventral ramus were retrogradely labelled with fast blue and diamidino yellow. In five animals that had received an embryonic graft 116 ± 16 cells were retrogradely labelled, and of these at least 15% were of graft origin, since they were positive for 5‐bromo‐2′‐deoxyuridine. In five animals that had their L4 ventral root reimplanted but did not receive a graft, only 12 ± 1.3 cells were retrogradely labelled. However, meaningful functional recovery could be achieved only if the regenerating axons of embryonic motoneurons found in the L4 ventral ramus were able to reverse the loss of force of muscles that had lost their innervation. This study shows that axons of embryonic motoneurons grafted into an adult rat spinal cord, as well as some axons of host origin, can be guided to denervated hindlimb muscles via reimplanted lumbar ventral roots. In normal rats ∼30 motor axons innervated the extensor digitorurn longus and 60 innervated the tibialis anterior via the L4 ventral root. In rats that did not receive a graft only 3.7 ± 1.2 axons reached the extensor digitorum longus and 3.5 ± 0.4 reached the tibialis anterior muscle via the implanted L4 ventral root. In animals that had an embryonic graft, 7.6 ± 0.5 axons innervated the extensor digitorum longus and 8.5 ± 0.5 reached the tibialis anterior muscle via the implanted root. In rats without a transplant the maximum tetanic tension elicited by stimulating the implanted L4 root was 16 ± 7 g for the extensor digitorum longus and 53 ± 36 g for the tibialis anterior muscle, whereas the corresponding muscles in animals that had an embryonic graft developed 82 ± 16 and 281 ± 95 g respectively. Thus it appears that the grafted motoneurons contributed to the innervation and functional recovery of the denervated muscles.

Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice.

The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella. Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration. Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time. Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation. Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells. CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation. The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged. By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level unit the Purkinje cells population had completely degenerated. Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

The Use of Embryonic Spinal Cord Grafts to Replace Identified Motoneuron Pools Depleted by a Neurotoxic Lectin, Volkensin

The viability of solid pieces of embryonic spinal cord grafted into adult spinal cords that had been depleted of motoneurons by a neurotoxic lectin, volkensin, was investigated. The possibility that the grafted neurons will extend their axons via the degenerated ventral roots and reinnervate the denervated muscles was also studied. Cells in the embryonic spinal cord were prelabeled with bromodeoxyuridine and grafted into the host cord 2 or 3 weeks after volkensin treatment. Six to 24 weeks later the fluorescent dyes Fast Blue and Diamidino Yellow were applied to the peripheral nerve or muscles which were expected to be reinnervated by neurons from the graft. The grafted cells were visualized by bromodeoxyuridine immunocytochemistry. Although the transplants survived, no neurons of graft origin reinnervated the muscles via the ventral roots of the host spinal cord. Therefore, in another series of experiments axons from grafted and host neurons were guided toward an implanted extensor hallucis longus (EHL) muscle via its motor nerve. The EHL muscle was removed from either healthy or volkensin-affected hindlimb of the host, placed paravertebrally, and the proximal end of its motor nerve inserted into the spinal cord at the site of the graft. In these experiments, neurons of graft origin reinnervated the paravertebrally placed muscle implant via the muscles own nerve. However, significantly fewer neurons were observed when volkensin-affected nerve-muscle implants were used. To establish whether grafted spinal cord neurons are necessary for reinnervation of skeletal muscles, in separate experiments embryonic neocortical tissue was transplanted into the motoneuron-depleted spinal cord. These grafts were connected to healthy nerve-muscle implants. In this case little reinnervation of the muscle occurred. These results suggest that, though volkensin-affected peripheral nerve is not a good conduit for regenerating axons, other inhibitory effects located in the CNS may be responsible for the failure of growing embryonic neurons to reach their peripheral targets via the existing anatomical routes. Experiments with grafted neocortical tissue indicate that good reinnervation of implanted muscles can only be achieved by using homologous embryonic tissue.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

Abstract Background: Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. Methods: Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2–12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. Results: Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2–4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Müller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. Conclusions: Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8–12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

Light microscopic histochemistry of the postnatal development and localization of carbonic anhydrase activity in glial and neuronal cell types of the rat central nervous system

SummaryThe postnatal expression of carbonic anhydrase (CA) activity in glia and neurons was investigated by a modified light microscopical method of Hansson. Strong CA activity was observed during the first postnatal week in ameboid microglia, clustering in the cingulum, the periaqueductal region, the roof of the lateral ventricles and the white matter of the cerebellum. The intensity of staining gradually decreased during the second week and finally disappeared. From the 9th postnatal day on, cerebellar Purkinje cells expressed strong CA activity, which completely disappeared by the end of the investigation period. CA staining of the oligodendrocytes and pericytes could be observed from the first postnatal day on. The present results raised the possibility that carbonic anhydrase activity may play a role in the regulation of the development and proliferation of some cell types, perhaps via intracellular pH changes.

Distribution of carbonic anhydrase activity in the rat central nervous system, as revealed by a new semipermeable technique

Results obtained with a new method provided evidence for the extraneuronal localization of CA and supports the idea that the enzyme is very widespread in non-neuronal cell types of the CNS. Most of these cells were considered to be oligodendroglia, but probably the astrocytes also contributed to the reactivity of the neuropil. The perineuronal CA activity observed in the spinal cord, brainstem, cerebellum, and hippocampus could be astrocytic in origin. Our observations concerning the widespread CA staining of the CNS vessels pointed to the possible functional significance of CA in the vessel wall. This activity could be due not only to pericytes but also to astrocytic processes. We have not found stained myelin sheats although biochemically the myelin contained the enzyme. Might be that our histochemical reaction was not sensitive to the membrane bound form of the CA. The fact that the reaction of the nucleoli did not disappear after acetazolamide treatment could be explained on the basis of binding of the cobalt-phosphate complex to the proteins of the nucleolus.

Expression and quantitative changes of carbonic anhydrase in developing neurones of rat central nervous system.

Postnatal changes in carbonic anhydrase activity were investigated in the islands of Calleja, which have been previously reported to contain the enzyme. Results obtained with a new modified method of Hansson provided further evidence for the distinction between the medial and lateral islands of Calleja. The enzyme was localized mainly in the nucleus and cytoplasm of granule cells without showing binding to any cytoplasmic organelle. No large neurons of the islands displayed carbonic anhydrase reactivity. The time course and rate of increase of carbonic anhydrase expression were different in the giant island of Calleja and lateral islands and this finding may strengthen the hypothesis regarding the medio‐lateral diversity of Callejas islands. On the other hand, at the end of the maturation process the granule cell complexes showed no significant difference in the proportion of carbonic anhydrase positive neurones. The almost equal rate of appearance of carbonic anhydrase reactive granule cells raises the possibility of a basic common role of both medial and lateral islets.

Neuronal carbonic anhydrase activity in the central nervous system of the rat: Light- and electron histochemical investigations of the islands of Calleja

Results obtained with a new semipermeable method provided evidence for the neuronal localization of carbonic anhydrase in the olfactory tubercle of the rat brain. Examination of serial coronal sections revealed the carbonic anhydrase content of the granule cells in the islands of Calleja. The enzyme was localized in the nucleus and the cytoplasm of these neurons and did not show binding to any of the intracellular organelles. The large neurons of the olfactory tubercle did not contain the enzyme. The enzyme activity of the granule cells was totally abolished by 10(-4) mol/l acetazolamide. Since, up to now, carbonic anhydrase activity has not been demonstrated with histochemical techniques in any central neurons, our results are the first indication of the presence of this enzyme in mammalian central neurons.