A comparison between chemical and gas hypoxia as models of global ischemia in zebrafish (Danio rerio).

BACKGROUND: Zebrafish models for neurovascular diseases offer new methods for elucidation of molecular pathways to tissue damage. External fertilization and high fecundity provide opportunities for transgenics and other forms of genetic manipulation that are more accessible than offered by mammalian models of disease. Furthermore, behavioral analyses of zebrafish allow for connection of molecular pathways to organismal outputs such as locomotion, learning, and memory. Unfortunately, a zebrafish model of hypoxia-ischemia has been slow to catch on, possibly due to hypoxia exposure protocols that are challenging to reproduce and result in high mortality. METHODS: In this study, we have introduced a predictable and simple method of hypoxia induction, the addition of sodium sulfite to aquarium water. The effects of this treatment on zebrafish locomotion were compared to those of zebrafish exposed to hypoxia induced by nitrogen gas bubbling, a method used in previous reports. RESULTS: We found that hypoxia induced by sodium sulfite significantly impaired locomotion in the hours following treatment, and its effects did not differ from those caused by nitrogen gas hypoxia. CONCLUSION: These results indicate that hypoxia by sodium sulfite represents an effective and easily reproducible method for the study of hypoxia-ischemia in zebrafish.

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research.

BACKGROUND: Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency. METHODS: To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). RESULTS: The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice. CONCLUSIONS: Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency.

Glial activation in the spinal ventral horn caudal to cervical injury.

Microglia and astrocytes play complex roles following spinal cord injury (SCI), contributing to inflammatory processes that both exacerbate injury and promote functional recovery by supporting neuro-protection and neuroplasticity. The crossed phrenic phenomenon (CPP) is an example of respiratory plasticity in which C(2) cervical hemisection (C(2)HS) strengthens crossed-spinal synaptic pathways to phrenic motor neurons ipsilateral to injury. We hypothesized that microglia and astrocytes are activated in the phrenic motor nucleus caudal and ipsilateral to C(2)HS, suggesting their potential for involvement in the CPP. To test this hypothesis, an incomplete cervical spinal hemisection (C(2) lateral injury; C(2)LI) was performed, and rats were allowed to recover for 1, 3, 14 or 28 days before collecting perfused spinal tissues. Microglia (via OX42) and astrocytes [via glial fibrillary acidic protein (GFAP)] were visualized with immunofluorescence microscopy in the C(4)-C(5) ventral horn, the region encompassing most of the phrenic motor nucleus. OX42-occupied fractional area ipsilateral to injury increased with C(2)LI (vs. sham) at 1 (12.5±1.8%, p<0.001), 3 (29.0±1.9%, p<0.001), 14 (26.1±3.1%, p<0.001) and 28 (19.2±2.0%, p<0.001) days post-C(2)LI. GFAP-occupied fractional area also increased with C(2)LI at 3 (24.4±3.2%, p<0.001) and 14 (16.8±8.3%, p=0.012) days, but not at 1 (6.2±3.9%, p=0.262) or 28 (10.6±3.9%, p=0.059) days post-C(2)LI. Thus, microglia and astrocytes are activated in the phrenic motor nucleus caudal to C(2)LI, suggesting that they play a role in functional deficits and/or recovery following spinal injury.

Lipopolysaccharide attenuates phrenic long-term facilitation following acute intermittent hypoxia.

Lipopolysaccharide (LPS) induces inflammatory responses, including microglial activation in the central nervous system. Since LPS impairs certain forms of hippocampal and spinal neuroplasticity, we hypothesized that LPS would impair phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) in outbred Sprague-Dawley (SD) and inbred Lewis (L) rats. Approximately 3h following a single LPS injection (i.p.), the phrenic response during hypoxic episodes is reduced in both rat strains versus vehicle treated, control rats (SD: 84 ± 7% vs. 128 ± 14% baseline for control, p < 0.05; L: 62 ± 10% vs. 90 ± 9% baseline for control, p < 0.05). At 60 min post-AIH, pLTF is also diminished by LPS in both strains: (SD: 22 ± 5% vs. 73.5 ± 14% baseline for control, p < 0.05; L: 18 ± 15% vs. 56 ± 8% baseline for control, p < 0.05). LPS alone does not affect phrenic burst frequency in either rat strain, suggesting that acute LPS injection has minimal effect on brainstem respiratory rhythm generation. Thus, systemic LPS injections and (presumptive) inflammation impair pLTF, a form of spinal neuroplasticity in respiratory motor control. These results suggest that ongoing infection or inflammation must be carefully considered in studies of respiratory plasticity, or during attempts to harness spinal plasticity as a therapeutic tool in the treatment of respiratory insufficiency, such as spinal cord injury.

Lysosomal release of cathepsins causes ischemic damage in the rat hippocampal slice and depends on NMDA-mediated calcium influx, arachidonic acid metabolism, and free radical production.

NMDA-mediated calcium entry and reactive oxygen species (ROS) production are well-recognized perpetrators of ischemic neuronal damage. The current studies show that these events lead to the release of the protein hydrolase, cathepsin B, from lysosomes 2 h following 5-min oxygen-glucose deprivation in the rat hippocampal slice. This release reflects a lysosomal membrane permeabilization (LMP) and was measured as the appearance of diffuse immunolabeled cathepsin B in the cytosol of CA1 pyramidal neurons. Necrotic neuronal damage begins after the release of cathepsins and is prevented by inhibitors of either cathepsin B or D indicating that the release of cathepsins is an important mediator of severe damage. There was an increase in superoxide levels, measured by dihydroethidium fluorescence, at the same time as LMP and reducing ROS levels with antioxidants, Trolox or N-tert-butyl-alpha-phenyl nitrone, blocked LMP. Both LMP and ROS production were blocked by an NMDA channel blocker (MK-801) and by inhibitors of mitogen-activated protein kinase kinase (U0126), calcium-dependent/independent phospholipases A2 (methyl arachidonyl fluorophosphonate) but not calcium-independent phospholipases A2 (bromoenol lactone) and cyclooxygenase-2 (NS398). A cell-permeant specific inhibitor of calpain (PD150606) prevented LMP, but not ROS production. It is concluded that LMP results in part from calcium-initiated and extracellular signal-regulated kinase-initiated arachidonic acid metabolism, which produces free radicals; it also requires the action of calpain.

A Bax-induced pro-oxidant state is critical for cytochrome c release during programmed neuronal death.

Bax is required for the apoptotic death of sympathetic neurons deprived of nerve growth factor (NGF). After NGF withdrawal, Bax translocates from the cytoplasm to the mitochondria of these cells and induces release of the proapoptotic protein cytochrome c. Here, we report that withdrawing NGF from mouse sympathetic neurons caused an increase of mitochondria-derived reactive oxygen species (ROS). Suppressing these ROS inhibited apoptosis. Bax deletion blocked death and prevented the ROS burst. Inducing a pro-oxidant state similar to that in NGF-deprived, wild-type cells caused cytochrome c release even in neurons lacking Bax. A similar ROS burst in cerebellar granule neurons undergoing apoptosis was also blocked by Bax deletion. These findings indicate that Bax lies upstream from increased ROS in NGF-deprived neurons and suggest that the Bax-induced ROS burst is both necessary and sufficient for cytochrome c redistribution in these cells.