Neuroprotection of grafted neurons with a GDNF/caspase inhibitor cocktail.

Transplantation of fetal ventral mesencephalic (VM) tissue shows great promise as an experimental therapy for patients with Parkinson's disease. However, cell survival in brain tissue grafts is poor, with survival rates of only 5-15%. We have utilized a combination of the caspase inhibitor bocaspartyl (OMe)-fluoromethylketone (BOC-ASP-CH2F) and glial cell line-derived neurotrophic factor (GDNF) to enhance survival of grafted dopamine neurons. The VM tissue was dissected from embryonic day 13-15 rat fetuses, incubated in different doses of BOC-ASP-CH2F and GDNF, and transplanted to the anterior chamber of the eye of adult rats. Growth of the tissue was assessed through the translucent cornea. Doses of 50 and 100 micromolar of the general caspase inhibitor appeared to have detrimental effects on mesencephalic tissue, while 20 micromolar had beneficial effects on overall transplant growth. A combination of the caspase inhibitor and GDNF appeared to have more prominent effects on cell survival as well as dopaminergic fiber density than either agent by itself. The transplants doubled in size when they were treated with a combination of BOC-ASP-CH2F and GDNF, and cell death markers were significantly reduced at both 48 h and 4-6 days postgrafting. This is, to our knowledge, the first combined approach using apoptotic blockers with trophic factors, and demonstrates a viable strategy for protection of developing neurons, since several different aspects of graft function may be addressed simultaneously.

Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hydroxydopamine lesion.

The neurotoxin 6-hydroxydopamine (6-OHDA) has been used extensively in animal models of Parkinson's disease. Typically, rodents develop severe unilateral movement deficiencies coupled with apomorphine-induced rotation behavior at least 1 week after an ipsilateral 6-OHDA lesion of the nigrostriatal dopamine (DA) system. The short-term morphological effects of 6-OHDA have not been determined in detail, however, and the exact process by which neurons die has not been elucidated. Thus, novel degenerative markers were used to determine the temporal pattern of acute phenotypic and degenerative alterations following a unilateral 6-OHDA injection into the medial forebrain bundle of adult rats. 6-Hydroxydopamine administration resulted in an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining as early as 6 hours postlesion. Staining for FluoroJade, a marker of neuronal degeneration, was evident at all time points examined but was maximal at 48 hours. Loss of tyrosine hydroxylase (TH) immunoreactivity began in axons at 6 hours, and progressed to cell bodies at later time points postlesion. Morphological examination of these neurons supported the conclusion of their death via apoptosis. Thus, whereas behavioral manifestations typically become evident 1 week or more following a 6-OHDA lesion, it is evident that nigral cell degeneration begins much earlier. This suggests multiple therapeutic possibilities, including the prevention of apoptosis, in affected neurons.

Glial cell line-derived neurotrophic factor is essential for postnatal survival of midbrain dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent trophic factors that have been identified for midbrain dopamine (DA) neurons. Null mutations for trophic factor genes have been used frequently for studies of the role of these important proteins in brain development. One problem with these studies has been that often only prenatal development can be studied because many of the knockout strains, such as those with GDNF null mutations, will die shortly after birth. In this study, we looked at the continued fate of specific neuronal phenotypes from trophic factor knockout mice beyond the time that these animals die. By transplanting fetal neural tissues from GDNF -/-, GDNF +/-, and wild-type (WT) mice into the brain of adult wild-type mice, we demonstrate that the continued postnatal development of ventral midbrain dopamine neurons is severely disturbed as a result of the GDNF null mutation. Ventral midbrain grafts from -/- fetuses have markedly reduced DA neuron numbers and fiber outgrowth. Moreover, DA neurons in such transplants can be "rescued" by immersion in GDNF before grafting. These findings suggest that postnatal survival and/or phenotypic expression of ventral mesencephalic DA neurons is dependent on GDNF. In addition, we present here a strategy for studies of maturation and even aging of tissues from trophic factor and other knockout animals that do not survive past birth.

Proliferative verrucous leukoplakia: report of two cases and a discussion of clinicopathology.

Proliferative verrucous leukoplakia (PVL) is a recently delineated but poorly recognized form of multifocal leukoplakia that is premalignant and of unproven origin. PVL generally presents as a simple benign form of hyperkeratosis that tends to spread and become diffuse. Although slow-growing, the disease is persistent and irreversible. Clinically, PVL often presents as an exophytic wart-like form of leukoplakia that appears to be resistant to nearly all forms of therapy. PVL of the oral cavity is best-defined as a continuum of oral epithelial disease with hyperkeratosis at one end of a clinical and microscopic spectrum and verrucous carcinoma or squamous cell carcinoma at the other. The microscopic findings associated with PVL are dependent on the stage of the disease and the adequacy of the biopsy. Microscopic findings can be markedly variable. PVL is a clinicopathologic disorder that includes the microscopic entity known as verrucous hyperplasia as a component of its histopathologic progression. This article reports on two cases of PVL, describes the clinicopathology of the disease process, and presents therapeutic and etiologic considerations.