Electrical preconditioning attenuates progressive necrosis and cavitation following spinal cord injury.

This study evaluates the influence of preconditioning and subsequent electrical stimulation on the formation of primary and secondary lesions following spinal cord injury in rats. The dorsal surface of the spinal cord at the T7 level was stimulated 24 h before right-side hemisection (500 Hz, 10 pulses/train, at an inter-train interval of 10 sec for 2 h). Stimulation was again administered immediately after injury and then every 24 h for 7 days. Preconditioning electrical stimulation of the spinal cord activated reactive astrocytes, then significantly attenuated edema, progressive necrosis, and cavitation, especially in the secondary cavity lesions (24 h, 1 week, and 3 weeks post-injury). Upregulation of glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity, a measure of reactive astrocytic response, were increased at 1 week after injury in the rats treated with electrical stimulation. These results suggest that preconditioning with electrical stimulation prevents the formation of secondary lesions after spinal cord injury. This beneficial effect may be related to the ability of electrical stimulation to attenuate trauma-induced cellular cascades.

Both MK801 and NBQX reduce the neuronal damage after impact-acceleration brain injury.

To understand the pathogenesis of diffuse axonal injury, we investigated the temporal and spatial profiles of neuronal degeneration in impact-acceleration injury in rats using Fluoro-Jade (FJ) staining. Impact-acceleration injury was produced in Wistar rats by the method described by Marmarou et al. with some modifications. Animals were sacrificed 1, 2, 7, 14, or 28 days after injury. Paraffin-embedded coronal sections were stained with HE or FJ, or analyzed immunohistochemically for GFAP or amyloid precursor protein (APP). FJ-positive degenerative neurons were found primarily in the dorsal brainstem and thalamus from 1 to 2 days following injury and these were associated with GFAP expression. However, FJ-positive cells were rarely found after 7 days. In all rats, significant expression of APP was observed primarily in the cingulum, cerebral peduncle and pontomedullary junction. FJ also stained these injured axons. Intrathecal administration of both NMDA and AMPA/kinate glutamate receptor antagonists MK-801 and NBQX, respectively, reduced the neuronal injury. NBQX showed more significant effects on axonal injury than MK-801. These observations indicate that not only axonal damage, but also primary neuronal damage occurs in this impact-acceleration injury model. It is also suggested that NBQX can act both directly on neuronal cells and white matter and that NMDA could have a significant protective effect against not only neuronal, but also axonal injury.

Fluorescence polarization of green fluorescence protein.

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60 degrees to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.