Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of speci®city for de®ning neuropathological cascades. Identi®cation of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Nonerythroid aII-spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase-3 cysteine proteases. As we have previously demonstrated, cleavage of aII-spectrin by calpain and caspase-3 results in accumulation of proteasespeci®c spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of aII-spectrin and aII-SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native aII-spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain-speci®c SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain-speci®c 145 kDa SBDP in CSF were 244%, 530% and 665% of sham-injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase-3-speci®c SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of aII-spectrin and aIISBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase-3.

High-resolution imaging of human autopsy tissues may improve our understanding of in vivo MRI findings, but interpretation is complicated because samples are obtained by immersion fixation following a postmortem interval (PMI). This study tested the hypotheses that immersion fixation and PMI's from 0 - 24 hours would alter the water relaxation and diffusion properties in rat cortical slice and spinal cord models of human nervous tissue. Diffusion data collected from rat cortical slices at multiple diffusion times (10 - 60 ms) and b-values (7 - 15,000 s/mm2) were analyzed using a two-compartment model with exchange. Rat spinal cords were characterized with standard diffusion tensor imaging (21 directions, b = 1250 s/mm2). Switching from perfusion- to immersion-fixation at 0-hrs PMI altered most MRI properties of rat cortical slices and spinal cords, including a 22% decrease in fractional anisotropy (P < 0.001). After 4 hrs PMI, cortical slice T1 and T2 increased 22% and 65% respectively (P < 0.001), transmembrane water exchange decreased 23% (P < 0.001) and intracellular proton fraction increased 25% (P = 0.002). After 6 hrs PMI, spinal cord white matter fractional anisotropy had decreased 38% (P < 0.001). MRI property changes were observed for PMIs up to 24 hours. The MRI changes correlated with protease activity and histopathological signs of autolysis. Thus, immersion fixation and/or even short PMIs (4-6 hours) altered the MRI properties of rat nervous tissue. This suggests comparisons between in vivo clinical MRI and MRI data from human autopsy tissues should be interpreted with caution.

Summary:Preclinical studies have identified numerous neuroprotective drugs that attenuate brain damage and improve functional outcome after cerebral ischemia. Despite this success in animal models, neuroprotective therapies in the clinical setting have been unsuccessful. Identification of biochemical markers common to preclinical and clinical cerebral ischemia will provide a more sensitive and objective measure of injury severity and outcome to facilitate clinical management and treatment. However, there are currently no effective biomarkers available for assessment of stroke. Nonerythroid ␣II-spectrin is a cytoskeletal protein that is cleaved by calpain and caspase-3 proteases to signature ␣II-spectrin breakdown products (␣II-SBDPs) after cerebral ischemia in rodents. This investigation examined accumulation of calpain-and caspase-3-cleaved ␣II-SBDPs in cerebrospinal fluid (CSF) of rodents subjected to 2 hours of transient focal cerebral ischemia produced by middle cerebral artery occlusion (MCAO) followed by reperfusion. After MCAO injury, full-length ␣II-spectrin protein was decreased in brain tissue and increased in CSF from 24 to 72 hours after injury. Whereas ␣II-SBDPs were undetectable in sham-injured control animals, calpain but not caspase-3 specific ␣II-SBDPs were significantly increased in CSF after injury. However, caspase-3 ␣II-SBDPS were observed in CSF of some injured animals. These results indicate that ␣II-SBDPs detected in CSF after injury, particularly those mediated by calpain, may be useful diagnostic indicators of cerebral infarction that can provide important information about specific neurochemical events that have occurred in the brain after acute stroke.

Oscillating gradient spin-echo (OGSE) pulse sequences have been proposed for acquiring diffusion data with very short diffusion times, which probe tissue structure at the subcellular scale. OGSE sequences are an alternative to pulsed gradient (PGSE) measurements, which typically probe longer diffusion times due to gradient limitations. In this investigation, a high-strength (6600 G/cm) gradient designed for small-sample microscopy was used to acquire OGSE and PGSE data in a rat hippocampal specimen at microscopic resolution. Measurements covered a broad range of diffusion times (TDeff=1.2–15.0 ms), frequencies (ω=67–1000 Hz) and b-values (b=0–3.2 ms/μm2). Variations in ADC with frequency and diffusion time provided microstructural information at a scale much smaller than the imaging resolution. For a more direct comparison of the techniques, OGSE and PGSE data were acquired with similar effective diffusion times. Measurements with similar TDeff were consistent at low b-value (b<1 ms/um2), but diverged at higher b-values. Experimental observations suggest that the effective diffusion time can be helpful in the interpretation of low b-value OGSE data. However, caution is required at higher b, where enhanced sensitivity to restriction and exchange render the effective diffusion time an unsuitable representation. Oscillating and pulsed gradient diffusion techniques offer unique, complementary information. In combination, the two methods provide a powerful tool for characterizing complex diffusion within biological tissues.

We hypothesized that custom-designed microemulsions would effectively scavenge compounds from bulk media. Pluronic-based oil-in-water microemulsions were synthesized that efficiently reduced the free concentration of the local anesthetic bupivacaine in 0.9% NaCl. Both the molecular nature and concentration of the constituents in the microemulsions significantly affected extraction efficiencies. Pluronic F127-based microemulsions extracted bupivacaine more efficiently than microemulsions synthesized using other Pluronic surfactants (L44, L62, L64, F77, F87, F88, P104). Extraction was markedly increased by addition of fatty acid sodium salts due to greater oil/water interface area, increased columbic interaction between bupivacaine and fatty acids sodium salt, and greater surface activity. These data suggest that oil-in-water microemulsions may be an effective agent to treat cardiotoxicity caused by bupivacaine or other lipophilic drugs.