Dose and chemical modification considerations for continuous cyclic AMP analog delivery to the injured CNS.

In this investigation, two cell-permeable synthetic analogs of cAMP, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, which are widely used to elevate intracellular cAMP levels under experimental conditions, were investigated for their ability to dose-dependently improve histological and functional outcomes following continuous delivery in two models of incomplete spinal cord injury (SCI). The cAMP analogs were delivered via osmotic minipumps at 1-250 mM through an indwelling cortical cannula or by intrathecal infusion for up to 4 weeks after either a T8 unilateral over-hemisection or a C2-3 dorsolateral quadrant lesion, respectively. In both SCI models, continuous db-cAMP delivery was associated with histopathological changes that included sporadic micro-hemorrhage formation and cavitation, enhanced macrophage infiltration and tissue damage at regions beyond the immediate application site; no deleterious or beneficial effect of agent delivery was observed at the spinal injury site. Furthermore, these changes were accompanied by pronounced behavioral deficits that included an absence of progressive locomotor recovery, increased extensor tone, paralysis, and sensory abnormalities. These deleterious effects were not observed in saline-treated animals, in animals in which the db-cAMP dose did not exceed 1 mM, or in those animals that received a high dose (250 mM) of the alternative cAMP analog, 8-bromo-cAMP. These results demonstrate that, for continuous intraparenchymal or intrathecal administration of cAMP analogs for the study of biological or therapeutic effects within the central nervous system (CNS), consideration of the effective concentration applied as well as the potential toxicity of chemical moieties on the parent molecule and/or their activity needs to be taken into account.

Adaptations in the walking pattern of spinal cord injured rats.

Walking ability is a measure of recovery used in many studies that test experimental strategies to treat injuries or diseases of the central nervous system (CNS) in animal models. A common measure in the rat animal model of thoracic spinal cord injury (SCI) is visual inspection and scoring of hind limb activity, which allows the documentation of movements associated with the recovery of locomotor function. In this study, we expand on previously documented visible changes in the locomotor pattern following SCI. The spontaneous recovery of locomotion in rats with thoracic SCIs of variable extent was evaluated using electromyographic (EMG) and kinematic analysis while rats walked on an elevated runway. Comparisons with pre-lesion walking sequences revealed changes in the kinematics and in the muscle activation pattern of various muscles, including enhanced fore limb extensor activity, possibly reflecting an increased contribution to propulsion, altered recruitment of back muscles inserting into the hip (possibly to support stepping movements), and elevated posture during stance, which may compensate for deficits in weight support. These changes were noted in spinal cord injured rats with varying degrees of impairment, including animals with no visually detectable deficit in open-field walking. In summary, the presented results demonstrate that spinal cord injured rats develop alternative locomotor patterns following SCI that cannot be discriminated by the use of qualitative visually based analysis, thus urging the use of quantitative outcome measures in assessing motor function after SCI.

Task specific adaptations in rat locomotion: runway versus horizontal ladder.

In walking quadrupeds the alternating activity pattern of antagonistic leg muscles and the coordination between legs is orchestrated by central pattern generating networks within the spinal cord. These networks are activated by tonic input from the reticular formation in the brainstem. Under more challenging conditions, such as walking on a horizontal ladder, successful locomotion relies upon additional context dependent input from pathways such as the cortico- and rubro-spinal tracts. In this study we used electromyographic and kinematic approaches to characterize the adaptations in the walking pattern in adult uninjured rats crossing a horizontal ladder. We found that the placement of a hind limb on a rung precisely followed the placement of the ipsilateral fore limb. This is different to normal walking where the hind limb is placed behind the position of the ipsilateral fore limb. The increased reach of the hind limbs is achieved by increased flexion of the hip and rotation of the pelvis during the swing phase. Electromyographic observations showed decreased burst duration in Tibialis anterior an ankle flexor muscle. Further changes in the muscle activity pattern were likely due to the reduced stepping frequency during ladder walking. Following a lesion of the dorsal column, containing major parts of the corticospinal tract, we found an increased number of stepping errors and changes in the stepping strategy. The step length of the fore limbs was reduced and the hind limbs were frequently positioned on rungs other than those occupied by the fore limb.

Neutrophils both reduce and increase permeability in a cell culture model of the blood-brain barrier.

This study was carried out to determine the effects that human neutrophils have on permeability across a model of the blood-brain barrier (BBB) formed by primary cultures of bovine brain microvessel endothelial cells (BBMEC). Transendothelial electrical resistance (TEER) was used to measure changes in permeability across BBMEC monolayers in a dual compartment system, during neutrophil interactions. When neutrophils (5 x 10(6)/ml) were applied to monolayers, TEER increased (permeability decreased). Adenosine was implicated, since the TEER increase was blocked by adenosine deaminase (1 U/ml) and the adenosine A2 receptor antagonist ZM 241385 (at 10(-6) M but not 10(-8) M, implicating A2B receptors). Oxygen free radicals were implicated as the TEER increase was blocked by combined catalase (100 U/ml) and superoxide dismutase (60 U/ml). When a gradient of the bacterial chemoattractant peptide formyl methionyl leucine phenylalanine (fMLP, 10(-7) M) was applied to neutrophils, the TEER decreased (permeability increased), concurrent with migration. When fMLP (10(-7) M) was added to the neutrophils, without migration, no change occurred. The TEER decrease was blocked by loading endothelium with the calcium buffer BAPTA (10 microM) and partially blocked by the serine protease inhibitor aprotinin (20 microg/ml). Measures to block the potential extracellular triggers heparin binding protein, glutamate, oxygen free radicals and binding to intercellular cell adhesion molecule-1 (ICAM-1) were ineffective. These data indicate that neutrophils both reduce and increase permeability in a cell culture model of the BBB, correlated to their proximity and migration through the endothelium. They explore the role of neutrophils in BBB breakdown, and the formation or amelioration of vasogenic cerebral edema.