Sex differences in a hypoxia model of preterm brain damage.

Male sex is a well-established risk factor for poor neurodevelopmental outcome after premature birth. The mechanisms behind this sex-related difference are unknown. The damage associated with prematurity can be mimicked in rodents by prolonged exposure to sublethal postnatal hypoxia. This chronic hypoxia leads to anatomical changes in mice that strongly resemble the loss of volume, decreased myelination, and ventriculomegaly seen in preterm newborns. However, no sex differences have been previously noted in this rodent model. We hypothesized that sex comparisons in hypoxic mice would show sex-related differences in brain volume and white matter loss in response to the same degree of hypoxic insult. Mice were placed in chronic sublethal hypoxia from postnatal day 3-11. Cortical, hippocampal, and cerebellar volumes and myelination indices were measured. We found that the male hippocampus, normally larger than the female, undergoes a greater volume loss compared with females (p < 0.05). Myelination, generally greater in males, was significantly disrupted by hypoxia in neonatal male forebrain. These results support the use of this rodent model to investigate the basis of sex-related susceptibility to brain damage and develop new sex-based neuroprotective strategies.

Characterization of a canine model of autosomal recessive retinitis pigmentosa due to a PDE6A mutation.

PURPOSE: To characterize a canine model of autosomal recessive RP due to a PDE6A gene mutation. METHODS: Affected and breed- and age-matched control puppies were studied by electroretinography (ERG), light and electron microscopy, immunohistochemistry, and assay for retinal PDE6 levels and enzymatic activity. RESULTS: The mutant puppies failed to develop normal rod-mediated ERG responses and had reduced light-adapted a-wave amplitudes from an early age. The residual ERG waveforms originated primarily from cone-driven responses. Development of photoreceptor outer segments stopped, and rod cells were lost by apoptosis. Immunohistochemistry demonstrated a marked reduction in rod opsin immunostaining outer segments and relative preservation of cones early in the disease process. With exception of rod bipolar cells, which appeared to be reduced in number relatively early in the disease process, other inner retinal cells were preserved in the early stages of the disease, although there was marked and early activation of Müller glia. Western blot analysis showed that the PDE6A mutation not only resulted in a lack of PDE6A protein but the affected retinas also lacked the other PDE6 subunits, suggesting expression of PDE6A is essential for normal expression of PDE6B and PDE6G. Affected retinas lacked PDE6 enzymatic activity. CONCLUSIONS: This represents the first characterization of a PDE6A model of autosomal recessive retinitis pigmentosa, and the PDE6A mutant dog shows promise as a large animal model for investigation of therapies to rescue mutant rod photoreceptors and to preserve cone photoreceptors in the face of a rapid loss of rod cells.

Development of bullwhip neurons in the embryonic chicken retina.

We have recently described large, unipolar neurons (named bullwhip cells) that regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken retina (Fischer et al. [2005] J. Neurosci. 25:10157-10166; [2006] J. Comp. Neurol. 496:479-494). There are only about 240 bullwhip cells in the entire retina, and these cells are easily identified by their unique morphology and immunoreactivity for glucagon, glucagon-like peptide 1 (GLP1), and substance P. The purpose of this study was to elucidate the development of bullwhip cells in the embryonic chicken retina. By using bromodeoxyuridine birth dating, we found that the bullwhip cells are generated very early during retinal development, between E4 and E5. Glucagon peptide was first detected in bullwhip cells at about E10, whereas substance P was not detected in the bullwhip cells until E15. Although glucagon peptide is not present during early stages of retinal development, we detected mRNA for glucagon receptor beginning at E7 and mRNA for GLP1 receptor at E5 through E14. Morphological differentiation of the bullwhip cells begins at about E14 and is completed by E18. The bullwhip cells are greatly overproduced, and nearly 80% of these cells undergo apoptotic cell death during late stages of embryonic development. The bullwhip cells that survive are those that project an axon-like process directly toward the CMZ; the cells that project in an inappropriate direction fail to survive. We conclude that cells fated to become bullwhip neurons are generated long before they begin to differentiate and that their survival depends on the orientation of their primary neurite.

Ultrasound-mediated gene transfer into neuronal cells.

A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.

Glucagon-expressing neurons within the retina regulate the proliferation of neural progenitors in the circumferential marginal zone of the avian eye.

Glucagon-expressing retinal amacrine cells have been implicated in regulating postnatal ocular growth. Furthermore, experimentally accelerated rates of ocular growth increase the number of neurons added to the peripheral edge of the retina. Accordingly, we assayed whether glucagon-expressing neurons within the retina regulate the proliferation of progenitors in the circumferential marginal zone (CMZ) of the postnatal chicken eye. We found that glucagon-containing neurites are heavily clustered within the CMZ at the peripheral edge of the retina. Many of these neurites originate from a cell type that is distinct from other types of retinal neurons, which we termed large glucagon-expressing neurons (LGENs). The LGENs are immunoreactive for glucagon and glucagon-like peptide 1 (GLP1), have a unipolar morphology, produce an axon that projects into the CMZ, and are found only in ventral regions of the retina. In dorsal regions of the retina, a smaller version of the LGENs densely ramifies neurites in the CMZ. Intraocular injections of glucagon or GLP1 suppressed the proliferation of progenitors in the CMZ, whereas a glucagon-receptor antagonist promoted proliferation. In addition, we found that glucagon, GLP1, and glucagon antagonist influenced the number of progenitors in the CMZ. We conclude that the LGENs may convey visual information to the CMZ to control the addition of new cells to the edge of the retina. We propose that glucagon/GLP1 released from LGENs acts in opposition to insulin (or insulin-like growth factor) to regulate precisely the proliferation of retinal progenitors in the CMZ.

Transitin, a nestin-related intermediate filament, is expressed by neural progenitors and can be induced in Müller glia in the chicken retina.

The purpose of this study was to test whether transitin, the avian homologue of nestin, is expressed by retinal progenitors in the developing and postnatal chicken. Because nestin has been widely used as a cell-distinguishing marker of neural progenitors in the mammalian nervous system, we expected to find transitin expressed specifically by the neural progenitors of the retina. In early stages of development, transitin is expressed by neural progenitors in the retina and by cells in the developing ciliary body. During later stages of development, transitin expression persists in differentiating Müller glia but is down-regulated by these cells as maturation proceeds. In the postnatal chick, transitin expression is restricted to neural progenitors at the peripheral edge of the retina. We found that the expression of transitin in mature Müller glia was induced by intraocular injections of insulin and fibroblast growth factor-2 (FGF2) but not by ciliary neurotrophic factor. In response to insulin and FGF2, the expression of transitin was induced in the nonpigmented epithelium (NPE) of the ciliary body. In the postnatal retina, acute retinal damage transiently induces transitin expression in Müller glia. We propose that the expression of transitin by retinal Müller glia and NPE cells in the postnatal animal represents a state of de-differentiation and a step toward becoming neurogenic progenitor cells. Taken together, our findings indicate that transitin is expressed by neural progenitors in the embryonic and postnatal chicken retina. However, transitin is not exclusively expressed by neural progenitors and is also expressed by non-neurogenic cells.

Different aspects of gliosis in retinal Muller glia can be induced by CNTF, insulin, and FGF2 in the absence of damage.

PURPOSE: In response to acute damage, Muller glia in the retina have been shown to dramatically alter their expression of filamentous proteins. Since damaged retinal cells are known to produce growth factors such as insulin-like growth factor (IGF), ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF), the altered expression of filaments in Muller glia in response to retinal damage may be induced by some of these factors. The purpose of this study was to assay whether growth factors influence the expression of filamentous proteins in Muller glia in the intact retinas of postnatal chickens. METHODS: We assayed for changes in expression levels of IGF-I, IGF-II, CNTF, FGF1, and FGF2 in N-methyl-D-aspartate(NMDA) damaged retinas by using quantitative PCR. In undamaged retinas, we assayed whether intraocular injections of insulin, CNTF, or FGF2 influenced glial expression of glial fibrillary acidic protein (GFAP), neurofilament, RA4, vimentin and beta3 tubulin by using immunocytochemistry on frozen sections. RESULTS: We demonstrated that levels of mRNA for IGF-II, FGF1, FGF2, and CNTF were increased in the postnatal chicken retina in response to neurotoxic damage. This was coincident with increased glial expression of GFAP and filamentous neuronal proteins. The combination of insulin and FGF2 caused postmitotic Muller glia to transiently increase their expression of vimentin and putative neuron specific filamentous proteins such as neurofilament, beta3 tubulin and RA4. By comparison, insulin or FGF2 alone had minor effects on glial expression of cytoskeletal proteins. Although neurofilament expression was not induced by CNTF, this growth factor stimulated Muller glia to express GFAP. CONCLUSIONS: We conclude that the phenotype of postmitotic Muller glia is plastic and can be regulated by retinal damage, and these damage induced changes in phenotype can be induced by exogenous growth factors in the absence of damage.

BMP4 and CNTF are neuroprotective and suppress damage-induced proliferation of Müller glia in the retina.

In response to acute damage, Müller glia in the chicken retina have been shown to be a source of proliferating progenitor-like cells. The secreted factors and signaling pathways that regulate this process remain unknown. The purpose of this study was to test whether secreted factors, which are known to promote glial differentiation during development, regulate the ability of Müller glia to proliferate and become retinal progenitors in response to acute damage in mature retina. We made intraocular injections of BMP4, BMP7, EGF, NGF, BDNF, or CNTF before or after a single, toxic dose of N-methyl-d-aspartate (NMDA) and assayed for proliferating progenitor-like cells within the retina. We found that injections of BMP4, BMP7, or CNTF, but not EGF, NGF, or BDNF, before NMDA treatment reduced the number of Müller glia that proliferated and gave rise to progenitor-like cells. CNTF and BMP4, but not NGF or BDNF, greatly reduced the number of cells destroyed by toxin treatment indicating that these factors protect retinal neurons from a severe excitotoxic insult. Injections of CNTF 5 days before NMDA treatment prevented neurotoxin-induced cell death and Müller glial proliferation, while injections of BMP4 had no protective effect. In addition, CNTF injected after NMDA treatment suppressed glial proliferation, while BMP4 did not. We conclude that BMP4 and CNTF, when applied before a toxic insult, act as neuroprotective agents and likely suppress the proliferative response of Müller glia to retinal damage by attenuating the retinal damage; protecting bipolar and amacrine neurons from NMDA-induced cell death. When applied after a toxic insult, CNTF suppressed glial proliferation independent of levels of retinal damage.

Copper/zinc superoxide dismutase attenuates neuronal cell death by preventing extracellular signal-regulated kinase activation after transient focal cerebral ischemia in mice.

Recent studies have revealed that activation of extracellular signal-regulated kinase (ERK) may contribute to apoptosis, a cell death process involved in oxidative stress. We examined phosphorylation of ERK1/2 and oxidative stress after transient focal cerebral ischemia (FCI) using transgenic (Tg) mice that overexpress copper/zinc superoxide dismutase (SOD1). The mice were subjected to 60 min of middle cerebral artery (MCA) occlusion by intraluminal suture blockade followed by 1, 4, and 24 hr of reperfusion. Immunohistochemistry and Western blot analysis showed that phospho-ERK1 was markedly increased in the cortex within the MCA territory at 1 hr of reperfusion (p < 0.01), followed by a decrease at 24 hr in wild-type mice. Double staining with phospho-ERK1/2 and neuron-specific nuclear protein showed that phospho-ERK1/2 was primarily expressed in neurons. In SOD1 Tg mice, phospho-ERK1/2 was prominently reduced compared with nonischemic controls, shown by immunohistochemistry. Western blot analysis confirmed a significant decrease in phospho-ERK1/2 1 hr after FCI in the ischemic cortex (p < 0.005). Apoptotic-related DNA fragmentation was reduced in the ischemic cortex of SOD1 Tg mice compared with wild-type mice using a cell death assay. These results suggest that phosphorylation of ERK1/2 may be involved in apoptosis or cell death after transient FCI and that SOD1 may attenuate apoptotic cell death mediated by the mitogen-activated protein kinase/ERK pathway.