Faculty Bibliography

Although recent studies indicate that use of a single global transverse relaxation time, T(2), per metabolite is sufficient for better than +/-10% quantification precision at intermediate and short echo-time spectroscopy in young adults, the age-dependence of this finding is unknown. Consequently, the age effect on regional brain choline (Cho), creatine (Cr), and N-acetylaspartate (NAA) T(2)s was examined in four age groups using 3D (four slices, 80 voxels 1 cm(3) each) proton MR spectroscopy in an optimized two-point protocol. Metabolite T(2)s were estimated in each voxel and in 10 gray and white matter (GM, WM) structures in 20 healthy subjects: four adolescents (13 +/- 1 years old), eight young adults (26 +/- 1); two middle-aged (51 +/- 6), and six elderly (74 +/- 3). The results reveal that T(2)s in GM (average +/- standard error of the mean) of adolescents (NAA: 301 +/- 30, Cr: 162 +/- 7, Cho: 263 +/- 7 ms), young adults (NAA: 269 +/- 7, Cr: 156 +/- 7, Cho: 226 +/- 9 ms), and elderly (NAA: 259 +/- 13, Cr: 154 +/- 8, Cho: 229 +/- 14 ms), were 30%, 16%, and 10% shorter than in WM, yielding mean global T(2)s of NAA: 343, Cr: 172, and Cho: 248 ms. The elderly NAA, Cr, and Cho T(2)s were 12%, 6%, and 10% shorter than the adolescents, a change of under 1 ms/year assuming a linear decline with age. Formulae for T(2) age-correction for higher quantification precision are provided

OBJECTIVE: Although most mild traumatic brain injury (mTBI) patients suffer any of several post-concussion symptoms suggestive of thalamic involvement, they rarely present with any MRI-visible pathology. The aim here, therefore, is to characterize their thalamic metabolite levels with proton MR spectroscopy (1H-MRS) compared with healthy controls. METHODS: T1-weighted MRI and multi-voxel 1H-MRS were acquired at 3 Tesla from 20 mTBI (Glasgow Coma Scale score of 15-13) patients, 19-59 years old, 0-7 years post-injury; and from 17 age and gender matched healthy controls. Mixed model regression was used to compare patients and controls with respect to the mean absolute N-acetylaspartate (NAA), choline (Cho) and creatine (Cr) levels within each thalamus. RESULTS: The mTBI-induced thalamic metabolite concentration changes were under +/- 13.0% for NAA, +/- 13.5% for Cr and +/- 18.8% for Cho relative to their corresponding concentrations in the controls: NAA: 10.08 +/- 0.30 (mean +/- standard error), Cr: 5.62 +/- 0.18 and Cho: 2.08 +/- 0.09 mM. These limits represent the minimal detectable differences between the two cohorts. CONCLUSION: The change in metabolic levels in the thalamus of patients who sustained clinically defined mTBI could be an instrumental characteristic of 'mildness'. 1H-MRS could, therefore, serve as an objective laboratory indicator for differentiating 'mild' from more severe categories of head-trauma, regardless of the presence or lack of current clinical symptoms

Work in rodents has shown that cultured retinal progenitor cells (RPCs) integrate into the degenerating retina, thus suggesting a potential strategy for treatment of similar degenerative conditions in humans. To demonstrate the relevance of the rodent work to large animals, we derived progenitor cells from the neural retina of the domestic pig and transplanted them to the laser-injured retina of allorecipients. Prior to grafting, immunocytochemical analysis showed that cultured porcine RPCs widely expressed neural cell adhesion molecule, as well as markers consistent with immature neural cells, including nestin, Sox2, and vimentin. Subpopulations expressed the neurodevelopmental markers CD-15, doublecortin, beta-III tubulin, and glial fibrillary acidic protein. Retina-specific markers expressed included the bipolar marker protein kinase Calpha and the photoreceptor-associated markers recoverin and rhodopsin. In addition, reverse transcription-polymerase chain reaction showed expression of the transcription factors Dach1, Hes1, Lhx2, Pax6, Six3, and Six6. Progenitor cells prelabeled with vital dyes survived as allografts in the subretinal space for up to 5 weeks (11 of 12 recipients) without exogenous immune suppression. Grafted cells expressed transducin, recoverin, and rhodopsin in the pig subretinal space, suggestive of differentiation into photoreceptors or, in a few cases, migrated into the neural retina and extended processes, the latter typically showing radial orientation. These results demonstrate that many of the findings seen with rodent RPCs can be duplicated in a large mammal. The pig offers a number of advantages over mice and rats, particularly in terms of functional testing and evaluation of the potential for clinical translation to human subjects. Disclosure of potential conflicts of interest is found at the end of this article.

Despite the increasing importance of the pig as a large animal model, little is known about porcine neural precursor cells. To evaluate the markers expressed by these cells, brains were dissected from 60-day fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor. Porcine neural precursors could be grown as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis using reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, microarrays, and flow cytometry, and results compared with data from analogous human forebrain progenitor cells. Cultured porcine neural precursors widely expressed neural cell adhesion molecule (NCAM), polysialic acid (PSA)-NCAM, vimentin, Ki-67, and Sox2. Minority subpopulations of cells expressed doublecortin, beta-III tubulin, synapsin I, glial fibrillary acidic protein (GFAP), and aquaporin 4 (AQP4) consistent with increased lineage restriction. A human microarray detected porcine transcripts for nogoA (RTN4) and stromal cell-derived factor 1 (SDF1), possibly cyclin D2 and Pbx1, but not CD133, Ki-67, nestin, or nucleostemin. Subsequent RT-PCR showed pig forebrain precursors to be positive for cyclin D2, nucleostemin, nogoA, Pbx1, vimentin, and a faint band for SDF1, whereas no signal was detected for CD133, fatty acid binding protein 7 (FABP7), or Ki-67. Human forebrain progenitor cells were positive for all the genes mentioned. This study shows that porcine neural precursors share many characteristics with their human counterparts and, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

PURPOSE: To use progenitor cells isolated from the neural retina for transplantation studies in mice with retinal degeneration. METHODS: Retinal progenitor cells from postnatal day 1 green fluorescent protein-transgenic mice were isolated and characterized. These cells can be expanded greatly in culture and express markers characteristic of neural progenitor cells and/or retinal development. RESULTS: After they were grafted to the degenerating retina of mature mice, a subset of the retinal progenitor cells developed into mature neurons, including presumptive photoreceptors expressing recoverin, rhodopsin, or cone opsin. In rho-/- hosts, there was rescue of cells in the outer nuclear layer (ONL), along with widespread integration of donor cells into the inner retina, and recipient mice showed improved light-mediated behavior compared with control animals. CONCLUSIONS: These findings have implications for the treatment of retinal degeneration, in which neuronal replacement and photoreceptor rescue are major therapeutic goals.

The goal of the present study was threefold: to determine whether viable human retinal progenitor cells (hRPCs) could be obtained from cadaveric retinal tissue, to evaluate marker expression by these cells, and to compare hRPCs to human brain progenitor cells (hBPCs). Retinas were dissected from post-mortem premature infants, enzymatically dissociated, and grown in the presence of epidermal growth factor and basic fibroblast growth factor. The cells grew as suspended spheres or adherent monolayers, depending on culture conditions. Expanded populations were banked or harvested for analysis by RT-PCR, immunocytochemistry, and flow cytometry. hBPCs derived from forebrain specimens from the same donors were grown and used for RT-PCR. Post-mortem human retinal specimens yielded viable cultures that grew to confluence repeatedly, although not beyond 3 months. Cultured hRPCs expressed a range of markers consistent with CNS progenitor cells, including nestin, vimentin, Sox2, Ki-67, GD2 ganglioside, and CD15 (Lewis X), as well as the tetraspanins CD9 and CD81, CD95 (Fas), and MHC class I antigens. No MHC class II expression was detected. hRPCs, but not hBPCs, expressed Dach1, Pax6, Six3, Six6, and recoverin. Minority subpopulations of hRPCs and hBPCs expressed doublecortin, beta-III tubulin, and glial fibrillary acidic protein, which is consistent with increased lineage restriction in subsets of cultured cells. Viable progenitor cells can be cultured from the post-mortem retina of premature infants and exhibit a gene expression profile consistent with immature neuroepithelial cells. hRPCs can be distinguished from hBPC cultures by the expression of retinal specification genes and recoverin.

Recent work with mammalian neural stem cells has highlighted the role of cytokine signaling in the proliferation and differentiation of these multipotent cells. While the responsiveness of neural progenitors to exogenously applied growth factors has been demonstrated in vivo as well as in vitro, little attention has been given to the production of cytokines by these cells. Here we use immunocytochemistry, RT-PCR, and ELISA to show that under standard growth conditions multipotent neural progenitor cells from humans express multiple cytokines including IL-1alpha, IL-1beta, IL-6, TGF-beta1, TGF-beta2, TNF-alpha, but not IL-2, IL-4, or IFN-gamma. Neural progenitor cells from rat and mouse express some, but not all, of these cytokines under similar conditions. While the function of cytokine expression by neural progenitor cells remains to be elucidated, these signaling molecules are known to be involved in neural development and may play a role in the activation of quiescent stem cells by a variety of pathological processes.