IGF-I and bFGF improve dopamine neuron survival and behavioral outcome in parkinsonian rats receiving cultured human fetal tissue strands.

To promote dopamine cell survival in human fetal tissue strands transplanted into immunosuppressed 6-OHDA-lesioned rats, we have preincubated tissue in insulin-like growth factor-I (IGF-I, 150 ng/ml) and basic fibroblast growth factor (bFGF, 15 ng/ml) in vitro for 2 weeks. Growth factor treatment did not affect the rate of homovanillic acid production in vitro but increased overall dopamine neuron survival in animals after transplant from 1240 +/- 250 to 2380 +/- 440 neurons (P < 0.05). Animals in the growth factor-treated group had a significantly greater reduction in methamphetamine-induced rotation (66%) compared to control transplants (30%, P < 0.05). We conclude that in vitro preincubation of human fetal tissue strands with IGF-I and bFGF improves dopamine cell survival and the behavioral outcome of transplants.

Co-grafts of muscle cells and mesencephalic tissue into hemiparkinsonian rats: behavioral and histochemical effects.

Extracts from skeletal muscle cell cultures have been shown to increase levels of the enzyme tyrosine hydroxylase (TH) and promote survival of different types of developing neurons in vitro. To determine the effect of muscle cell co-grafts on the survival of dopamine neurons in a rat model of Parkinson's disease, we transplanted an embryonic day (ED)-15 rat mesencephalic cell suspension alone or with neonatal muscle cells into 6-hydroxydopamine (6-OHDA) denervated rat striatum. In parallel experiments conducted in vitro, we cultured ED-15 rat mesencephalon or rat striatum in conditioned medium from neonatal rat muscle cultures (MC-CM). Our results showed that: (A) in vitro, MC-CM increased the number of TH-immunoreactive (TH-IR) neurons in embryonic mesencephalic cultures but did not induce expression of TH in embryonic striatal cultures; (B) in vivo, animals with co-grafts of muscle cells and ED-15 mesencephalon had more TH-IR in the grafted striatum compared to animals that received mesencephalic cells grafts alone, although the graft-induced reversal of circling behavior in response to methamphetamine was the same in both transplanted groups; and (C) grafts of muscle cells alone did not induce TH-IR in the denervated striatum and did not reduce methamphetamine-induced circling. These findings suggest that in vivo, neonatal muscle cells secrete factors that promote survival and/or outgrowth of fetal midbrain dopamine cells and improve the levels of TH-IR in grafted striatum.

Strands of embryonic mesencephalic tissue show greater dopamine neuron survival and better behavioral improvement than cell suspensions after transplantation in parkinsonian rats.

The success of embryonic neural transplants as a treatment for patients with Parkinson's disease has been limited by poor survival of transplanted dopamine neurons. To see if a new partially intact tissue preparation method improves survival, we have developed a technique for extruding embryonic tissue into strands. We expected this method to reduce cell damage and improve transplant survival as well as provide improved tissue delivery. We have compared transplants of tissue strands with mechanically dispersed suspensions of embryonic day 15 rat ventral mesencephalon. Tissue from ventral mesencephalon was transplanted into a single site in dopamine denervated striatum of unilateral 6-hydroxydopamine (6-OHDA) lesioned rats. To evaluate the effects of striatal cografts and growth factors on dopamine cell survival, dispersed mesencephalic cells were cotransplanted with dispersed striatal cells. Another group had dispersed mesencephalic cells cotransplanted with striatal cells incubated in the cold for 2 h with glial cell line-derived neurotrophic factor (GDNF, 100 ng/ml), insulin-like growth factor-I (IGF-I, 1500 ng/ml), and basic fibroblast growth factor (bFGF, 150 ng/ml). Behavioral improvement was assessed monthly by changes in methamphetamine-induced rotational behavior. Animals were sacrificed after 3 months, and dopamine neurons were identified by tyrosine hydroxylase (TH) immunohistochemistry. Transplants of tissue strands produced better dopamine neuron survival and led to more robust behavioral restoration than did cell suspensions even when suspensions were supported with cografts of striatal cells or pretreatment with growth factors.

Growth factors improve immediate survival of embryonic dopamine neurons after transplantation into rats.

Embryonic dopamine neurons survive poorly after transplant into models of Parkinson's disease, possibly due to programmed cell death (apoptosis). Apoptosis in cultured dopamine neurons can be reduced by growth factors such as glial cell line-derived neurotrophic factor (GDNF) or a combination of insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF). To improve the survival of dopamine neurons in grafts, strands of E15 rat ventral mesencephalon were pretreated with a combination of GDNF, IGF-I, and bFGF and then transplanted into 6-hydroxydopamine-lesioned rats. In control animals, only 32% of dopamine neuron profiles survived the first 24 h after transplant. Growth factor pretreatment increased survival to 49% on day 1. Growth factors reduced the apoptotic rate of transplanted cells, just as they had in the previous in vitro experiments. Apoptotic nuclear morphology was observed in the transplanted dopamine neurons. We conclude that the majority of transplanted dopamine neurons die in grafts within the first 24 h after transplant, most likely by an apoptotic mechanism. Prevention of apoptosis with anti-apoptotic agents may improve the viability of dopamine neurons grafted for Parkinson's disease.

Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

Spontaneous myopathy in the SJL/J mouse: pathology and strength loss.

Myopathy has been found to develop spontaneously in 100% of SJL/J mice between 6 and 8 months of age. Extent of muscular involvement and mouse strength were quantified in SJL/J mice and Balb/c control mice 2-16 months old. Muscle from young SJL/J mice exhibited histopathological abnormalities and occasional inflammatory infiltrate. By 6 months, 78% of SJL/J mice had developed active myopathy. By 8 months, all SJL/J mice examined had active disease with a mean of 12.9% of muscle fibers affected. Replacement of muscle fibers by fat and/or collagen began at 10 months and was pronounced by 14 months. Significant decreases in strength scores (total body pulling force) at 6 months and 10 months of age reflected the onset of active myopathy and the onset of muscle degeneration, respectively. The spontaneous onset and 100% incidence of myopathy in the SJL/J mouse line should provide a useful model for idiopathic myopathy.

Glutamine synthetase: glial localization in brain.

Light microscopy immunohistochemical techniques were used to examine the distribution of glutamine synthetase in rat brain. Glutamine synthetase was found to be localized in the glial cells. Neuronal cell bodies, endothelial cells, and choroid epithelium contained no enzyme. The findings indicate that glia have a crucial role in glutamic acid, gamma-aminobutyric acid, and ammonia metabolism in brain.