Intrathecal cyclosporin prolongs survival of late-stage ALS mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood-brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.

Cyclosporine-A enhances choline acetyltransferase immunoreactivity in the septal region of adult rats.

Cyclosporine-A (CsA) is the primary anti-rejection drug used for organ and neural transplantation therapy. In addition to its immunosuppressive action, CsA has been recently shown to exert neuroprotective and neurotrophic effects in the central nervous system when able to cross the blood-brain barrier. Postulated mechanisms for these CsA-induced beneficial effects include the drug's powerful inhibition of the calcium-dependent phosphatase calcineurin (CN) and blockade of the assembly of the mitochondrial permeability transition pore. We report here, for the first time, that adult Wistar rats treated with CsA (10 mg/kg per day, i.p. for 9 days) displayed significantly reduced septal CN expression in combination with enhanced levels of septal choline acetyltransferase (ChAT) immunoreactivity as compared to controls. The observed enhancement of septal ChAT immunoreactivity suggests potential therapeutic utility of CsA for brain disorders characterized by alterations of the cholinergic system.

Role and mechanisms of secondary mitochondrial failure.

Ischemia is accompanied by mitochondrial dysfunction, as assessed by measurements of mitochondrial respiratory activities in vitro. Following brief periods of ischemia, mitochondrial function is usually normalized during reperfusion. However, particularly after ischemia of longer duration, reperfusion may be accompanied by secondary mitochondrial failure. After short periods of ischemia this is observed in selectively vulnerable areas and, after intermediate to long periods of ischemia, in other areas as well. However, it has remained unsettled if the mitochondrial dysfunction is the result or the cause of cell death. Although it has been commonly assumed that such failure is secondary to cell injury by other mechanisms, recent results suggest that mitochondrial dysfunction may be the cause of cell death. Indirect evidence for this postulate is provided by experiments showing that cyclosporin A (CsA), when allowed to cross the blood-brain barrier, is a potent neuroprotectant. CsA is a virtually specific blocker of the mitochondrial permeability transition (MPT) pore, a voltage-gated channel allowing molecules and ions with a mass < 1500 Daltons to pass the inner mitochondrial membrane. Experiments on isolated cells in vitro demonstrate that cell calcium accumulation or oxidative stress triggers the assembly of an MPT pore, which leads to collapse of the mitochondrial membrane potential, to ATP hydrolysis, to enhanced production of reactive oxygen species (ROS), and to cell death. The beneficial effect of CsA could thus be related to its ability to block the MPT pore. Longer periods of ischemia, such as occurs after transient middle cerebral artery (MCA) occlusion, lead to pan-necrotic lesions (infarction). In the rat, recirculation following 2 h of MCA occlusion leads to partial normalization of the bioenergetic state but this is followed within 4-6 h by secondary bioenergetic failure. The latter seems unrelated to blockade of the microcirculation, but correlates to secondary mitochondrial failure. The brain damage incurred is ameliorated by the spin trap alpha-phenyl-N-butyl nitrone (PBN) and by the immunosuppressant FK506 even when given 1-3 h after the start of recirculation. The two drugs also prevent the secondary mitochondrial failure during early recirculation, suggesting that such failure is pathogenetically important. Probably, though, the mitochondrial dysfunction involves not only the assembly of an MPT pore but also other mechanisms. Since recirculation is associated with release of mitochondrial proteins it is not unlikely that such proteins, e.g. cytochrome c, trigger cascades of events leading to cell death.6.

Suppression of kindling epileptogenesis in rats by intrahippocampal cholinergic grafts.

Selective immunolesioning of the basal forebrain cholinergic system by 192 IgG-saporin, which leads to a dramatic loss of the cholinergic innervation in cortical and hippocampal regions, facilitates the development of hippocampal kindling in rats. The aim of the present study was to explore whether grafted cholinergic neurones are able to reverse the lesion-induced increase of seizure susceptibility. Intraventricular 192 IgG-saporin was administered to rats which 3 weeks later were implanted with rat embryonic, acetylcholine-rich septal-diagonal band tissue ('cholinergic grafts') or cortical tissue/vehicle ('sham grafts') bilaterally into the hippocampal formation. After 3 months, the grafted animals as well as non-lesioned control rats were subjected to daily hippocampal kindling stimulations. In the animals with cholinergic grafts, which had reinnervated the hippocampus and dentate gyrus bilaterally, there was a marked suppression of the development of seizures as compared with the hyperexcitable, sham-grafted rats. This effect was significantly correlated to the density of the graft-derived cholinergic innervation of the host hippocampal formation. The kindling rate in the rats with cholinergic grafts was similar to that in non-lesioned controls. These results provide further evidence that the intrinsic basal forebrain cholinergic system dampens kindling epileptogenesis and demonstrate that this function can be exerted also by grafted cholinergic neurones.

Tirilazad mesylate improves survival of rat and human embryonic mesencephalic neurons in vitro.

The survival rate of embryonic dopamine (DA) neurons after transplantation to the striatum is only 5-20%. Therefore, mesencephalic tissue from several donors needs to be implanted in a parkinsonian patient to induce a therapeutic improvement. Lazaroids are a group of neuroprotective compounds which inhibit lipid peroxidation. Previously, two lazaroids (U-74389G and U-83836F) have been found to improve the survival of both cultured and grafted rat DA neurons. The only lazaroid approved for human use is tirilazad mesylate. The objective of the present study was to explore the effects of tirilazad mesylate on DA neuron survival in cultures of rat ventral mesencephalon and its capacity to promote the in vitro cell viability of embryonic rat and human mesencephalic tissue, treated and dissociated in the same way as in clinical trials. After 7 days in vitro, the number of tyrosine hydroxylase-immunopositive, presumed DA neurons was 140% higher in rat cultures treated with 0.3 microM tirilazad mesylate than that in control cultures. Rat and human cell suspensions supplemented with tirilazad mesylate maintained a high degree of viability for several hours longer than control suspensions. These results indicate that tirilazad mesylate promotes the survival of both rat and human embryonic mesencephalic neurons in vitro. Tirilazad mesylate can be administered clinically and may become a useful tool for increasing survival of grafted DA neurons in patients, thereby reducing the needed quantity of human donor tissue.

Effects of cholinergic denervation on seizure development and neurotrophin messenger RNA regulation in rapid hippocampal kindling.

Intraventricular 192 IgG-saporin was used to induce a selective lesion of basal forebrain cholinergic neurons in rats. When subjected to 40 rapid hippocampal kindling stimulations with 5-min intervals, these animals exhibited increased number of generalized seizures and a higher mean seizure grade in response to the first five stimulations, and required fewer stimuli to develop focal behavioural seizures, as compared to non-lesioned rats. In contrast, both groups showed similarly enhanced responsiveness when test stimulated four weeks later. Using in situ hybridization, cholinergic denervation was found to cause a significant decrease of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex, whereas gene expression for nerve growth factor, neurotrophin-3, and TrkB and TrkC was unchanged. Four weeks after rapid kindling stimulations, basal levels of brain-derived neurotrophic factor messenger RNA in the dentate granule cells were restored to normal in the lesioned rats, whereas neurotrophin-3 messenger RNA levels were decreased. No differences in the seizure-evoked levels of neurotrophin and Trk messenger RNAs were detected, except in the dentate granule cell layer, which had significantly higher brain-derived neurotrophic factor messenger RNA expression in the lesioned animals at 2 h. In conclusion, the basal forebrain cholinergic system (i) dampens the severity of recurring seizures induced by rapid hippocampal kindling stimulations, but has no effect on the subsequent delayed phase of epileptogenesis; and (ii) exerts a tonic stimulation of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex. The findings also indicate that the cholinergic lesion does not affect neurotrophin and Trk gene expression after recurring seizures, and that the kindling process leads to long-term changes in basal brain-derived neurotrophic factor and neurotrophin-3 messenger RNA levels in the denervated animals.

Regulation of neuronal nitric oxide synthase mRNA levels in rat brain by seizure activity.

The regulation of neuronal nitric oxide synthase (NOS) mRNA levels during kindling epileptogenesis in the rat brain was investigated using in situ hybridization. Following 40 rapidly recurring seizures evoked by hippocampal stimulations, NOS mRNA expression decreased by 56% in the dentate granule cell layer (maximum at 2 h) and increased by 420,105 and 1260% in the CA1 and CA3 pyramidal layers and piriform cortex, respectively (maximum at 12-24 h). Gene expression had returned to control levels after one week. The presumed alterations of nitric oxide production, following the changes in NOS mRNA shown here, may modulate synaptic function during kindling development, and could influence neuronal vulnerability after epileptic insults.

New insight on the factors orienting the axonal outgrowth of grafted Purkinje cells in the pcd cerebellum.

Despite Purkinje cell replacement, leading to the repair of the cortical circuit of the pcd mouse cerebellum grafted with E12 cerebellar primordium, the reestablishment of the corticonuclear projection only occurs for some Purkinje cells and in a small percentage of grafted mice. In order to assess the importance of: (1) competition between host and grafted deep nuclei, and (2) the distance between the implants and the host deep nuclei, new grafted experiments have been performed. In the latter, solid grafts were taken from E13 or E14 donor embryos after removal of the region containing the postmitotic deep nuclear neurons, and randomly positioned at various cerebellar depths. With cortical implants, the absence of donor nuclear neurons is not sufficient to allow the axons of the grafted Purkinje cells that have invaded the host molecular layer to escape the confinement of this layer. The molecular/granular layer interface appears as an almost impassable obstacle, and the granule cell layer as a nonpermissive milieu. With grafts located between the host deep nuclei and the 4th ventricle (deep grafts), the grafted Purkinje cells project massively to the host nuclei, but they are unable to leave the implant and, therefore, they are not integrated in the deficient cortical circuit. Finally, when the grafts positioned in the central white matter (intermediate grafts) disrupt the integrity of the host granule cell layer, some of the grafted Purkinje cells invade the host molecular layer, while most of them remain within the implant. Some axons of the cortically integrated Purkinje cells, using the nearby graft as a bridge, seem able to innervate the host deep nuclei. The latter, in addition, receive a massive projection from the nonintegrated Purkinje cells. These results emphasize the ability of grafted Purkinje cells to specifically innervate their target host neurons, when either there is proximity, or when a permissive microenvironment for their axonal outgrowth is created by embryonic grafted cortical cerebellar neurons, filling the gap between the molecular layer and the deep nuclei of the host.

Parasites of South African wildlife. VI. Helminths of blue duikers, Cephalophus monticola, in Natal.

The helminths of 3 blue duikers, Cephalophus monticola, from 3 nature reserves in Natal were collected, counted and identified. Taenia hydatigena larvae, a race of Cooperia rotundispiculum, gongylonema angistris, Trichostrongylus falculatus and Trichostrongylus rugatus appear to be new parasite records for blue duikers in South Africa.

Parasites of South African wildlife. III. Helminths of common reedbuck, Redunca arundinum, in Natal.

Twenty-six common reedbuck, Redunca arundinum, were shot in pairs at monthly intervals for 13 consecutive months in the Himeville region of Natal. Ten nematode species, 2 cestodes and 1 trematode were recovered from these animals. Cooperia yoshidai was both the most numerous and most prevalent worm and peak burdens occurred during summer. Thirty-one reedbuck, killed at different intervals in various localities within the St. Lucia Reserve, harboured between 4 and 11 nematode species, 1 cestode and 1 trematode. With the exception of 4 reedbuck shot during January 1987, in which Haemonchus contortus was the most abundant worm, C. yoshidai was again both the most abundant and most prevalent worm. Peak burdens of this nematode occurred during autumn to spring. The helminths of 5 impala, Aepyceros melampus, also shot in the St. Lucia Reserve were examined. Some of the worm species of impala were also found in the reedbuck from the same locality and the helminths of the 2 antelope species are compared. An amended list, which includes several new records of the parasites of common reedbuck in South Africa is provided.

Parasites of domestic and wild animals in South Africa. XXIV. Arthropod parasites of bushbuck and common duiker in the Weza State Forest, Natal.

One bushbuck, Tragelaphus scriptus, and 1 common duiker, Sylvicapra grimmia, were shot each month from May 1983 to May 1984 in the Weza State Forest, Natal, i.e. a total of 13 animals of each species. The bushbuck were infested with 8 ixodid tick species, 2 louse species and a louse-fly species. The common duiker harboured 7 tick species and 2 louse species. Ticks of the genus Ixodes were the most numerous and prevalent on both antelope species, but no pattern of seasonal abundance was evident. Although only small numbers were recovered, adult Haemaphysalis aciculifer were present from September to February, nymphs of Rhipicephalus appendiculatus from May to September, and adult Rhipicephalus lunulatus from December to March. The louse-fly, Lipoptena paradoxa, was recovered from some of the bushbuck from October to May.

Arthropod parasites of common reedbuck, Redunca arundinum, in Natal.

Twenty-five common reedbuck, Redunca arundinum, from the Himeville region, 21 from the Eastern Shores Nature Reserve, 4 from the Charter's Creek Nature Reserve and 2 from the St Lucia Game Park, Natal were examined for arthropod parasites. The reedbuck from Himeville were infested with 4 ixodid tick species, those from the Eastern Shores with 7 species and those from Charter's Creek and St Lucia with 6 species. Rhipicephalus evertsi evertsi was the only tick common to the 4 localities. The lice Damalinia reduncae and Linognathus fahrenholzi were present on the reedbuck from each locality. In addition 3 red duiker, Cephalophus natalensis, and 2 bushbuck, Tragelaphus scriptus, from the Charter's Creek Nature Reserve plus 2 impala, Aepyceros melampus, from the St Lucia Game Park were examined for ixodid ticks. The red duiker were infested with 3 tick species and the bushbuck and impala with 4 each.

Parasites of South African wildlife. I. Helminths of bushbuck, Tragelaphus scriptus, and grey duiker, Sylvicapra grimmia, from the Weza State Forest, Natal.

Thirteen bushbuck, Tragelaphus scriptus, and 13 grey duikers, Sylvicapra grimmia, were culled in the Weza State Forest, Natal, from May 1983-May 1984. The maximum number of worms recovered from a single bushbuck was 393. Of the total number of worms recovered from all the bushbuck, Paracooperia devossi were the most numerous, followed by Ostertagia harrisi, a Cooperia sp. and Haemonchus vegliai. P. devossi and O. harrisi were more numerous during winter and the Cooperia sp. more numerous during summer. Grey duiker harboured from 0-230 worms. The same Cooperia sp. as was recovered from the bushbuck and Trichostrongylus axei were the most numerous. The larvae of Taenia hydatigena were recovered from 5 duikers. The Cooperia sp. was more numerous during summer and T. axei during winter, the remaining worms showing no seasonal pattern of abundance. Amended lists of the helminth parasites found in these antelope in the Republic of South Africa are provided.

The helminths of various antelope species from Natal.

Helminth parasites were collected from 2 bushbuck, Tragelaphus scriptus, 2 red duiker, Cephalophus natalensis, 1 oribi, Ourebia ourebi, and 4 reedbuck, Redunca arundinum, that died or were culled in various parts of Natal. One trematode genus, 1 cestode genus and 12 nematode species were recovered. Haemonchus contortus. Ostertagia harrisi, Trichostrongylus capricola, Trichostrongylus vitrinus, Cooperia rotundispiculum and Setaria scalprum are new parasite records for the red duiker. Trichostrongylus colubriformis is a new parasite record for the oribi and Longistrongylus schrenki, Trichostrongylus falculatus, Trichostrongylus colubriformis and Dictyocaulus viviparus are recorded from the reedbuck for the first time. An unidentified paramphistome was also recovered from the reedbuck.

Vitamin B12 and its binding proteins in the serum of some wild game species.

The concentration of vitamin B12 and its binding proteins was measured in the impala, nyala, wildebeest, zebra, bushpig, warthog, and rhino, all existing in their natural state. Marked differences were found between some species. The variations observed are probably true species differences, unrelated to environmental factors.