Regional distribution of fluoro-jade B staining in the hippocampus following traumatic brain injury.

Fluoro-Jade B (FJB) is an anionic fluorescein derivative that has been reported to specifically stain degenerating neurons. We were interested in applying FJB staining in a well-characterized model of traumatic brain injury (TBI) in order to estimate the total number of neurons in different regions of the hippocampus that die after a mild or moderate injury. Rats were subjected to a mild or moderate unilateral cortical contusion (1.0- or 1.5-mm displacement from the cortical surface) with a controlled cortical impacting device. Animals were allowed to survive for 1, 2, or 7 days and the total number of FJB-positive neurons in hippocampal areas CA1, CA3, and the dentate gyrus granule layer was estimated using sterological methods. The region that had the highest number of FJP-positive neurons after TBI was the dentate gyrus. In both 1- and 1.5-mm injuries, FJB-positive granule cells were observed throughout the rostro-caudal extent of the dentate. In contrast, labeled pyramidal neurons of area CA3 were most numerous after the 1.5-mm injury. The area that had the fewest number of FJB-labeled cells was area CA1 with only scattered neurons seen in the 1.5-mm group. In both injury groups and in all hippocampal regions, more FJB-positive neurons were seen at the earlier times post injury (1 and 2 days) than at 7 days. FJB appears to be a reliable marker for neuronal vulnerability following TBI.

Nicotine attenuates morphological deficits in a contusion model of spinal cord injury.

Protection against the progression of secondary injury appears to be an effective therapeutic strategy in spinal cord injury (SCI). Evidence indicates that nicotine can induce potent neuroprotective effects against injury to spinal cord neurons. Therefore, the present study was focused on the effects of nicotine on the behavioral and morphological recovery associated with SCI. Adult male Long-Evans rats were subjected to a moderate contusion model of SCI and received subcutaneous injections of nicotine for 14 days at the dose of 0.35 or 7 mg/kg/day. The rats were examined using the BBB locomotor rating scale for 6 weeks. At the end of the BBB recording, spinal cords were examined for the volumetric tissue sparing of gray and white matters. All SCI rats demonstrated a loss of hindlimb function followed by a recovery phase that peaked at 2-3 weeks after the trauma. Compared to untreated SCI rats, chronic nicotine administration appeared to improve the recovery of the locomotor functions. Indeed, nicotine-treated animals scored consistently higher on the BBB scale indicating that the treatment altered animal behavior. However, when taking under consideration correction factors for multiple comparisons, these data did not reach significance at overall experimental levels of significance 0.05. Nevertheless, nicotine administration was effective in sparing tissue at injury epicenter and a lower dose of nicotine also resulted in significant sparing of white matter of the injured spinal cord. These results suggest that agonists of neuronal nicotinic receptors can be attractive candidates for SCI therapy.

Cleaved-tau: a biomarker of neuronal damage after traumatic brain injury.

Previous studies from our laboratory indicate that traumatic brain injury (TBI) in humans results in proteolysis of neuronally-localized, intracellular microtubule associated protein (MAP)-tau to produce cleaved tau (C-tau). The present study evaluated the utility of C-tau to function as a biomarker of neuronal injury and as a biomarker for evaluating neuroprotectant drug efficacy in a controlled cortical impact model of rat TBI. Brain C-tau was determined in rats subjected to controlled cortical impact-induced mild, moderate or severe levels of TBI. A significant severity-dependent increase in C-tau levels was observed in the cortex and hippocampus (1.5-8-fold) of TBI rats compared to shams 72 h after impact. C-tau rat brain and serum time course was determined by measuring levels at 0.25, 6, 24, 48, 72 and 168 h after TBI. A significant time-dependent increase in C-tau levels was observed in ipsilateral cortex (5-16-fold) and hippocampus (2-40-fold) compared to sham animals. C-tau levels increased as early as 6 h after TBI with peak C-tau levels observed 168 h after injury. Elevated brain C-tau levels were associated with TBI-induced tissue loss, which was histologically determined. The effect of cyclosporin-A (CsA), previously demonstrated to be neuroprotective in rat TBI, on brain C-tau levels was examined. CsA (20 mg/kg i.p., 15 min and 24 h after TBI) significantly attenuated the TBI-induced increase in hippocampal C-tau levels observed in vehicle-treated animals confirming CsA's neuroprotectant effect. CsA treatment also lowered ipsilateral cortical C-tau levels, although it did not reach statistical significance. CsA's neuroprotectant effect was confirmed utilizing histologic measures of TBI-induced tissue loss. In addition, serum C-tau levels were significantly increased 6 h after TBI but not at later time points. These results suggest that C-tau is a reliable, quantitative biomarker for evaluating TBI-induced neuronal injury and a potential biomarker of neuroprotectant drug efficacy in the rat TBI model. Serum data suggests that C-tau levels are dependent both on a compromised blood-brain barrier as well as release of TBI biomarkers from the brain, which has implications for the study of human serum TBI biomarkers.

Fluoro-jade B stains quiescent and reactive astrocytes in the rodent spinal cord.

In an attempt to label dying neurons in the injured spinal cord, we used the novel fluorescein derivative Fluoro-Jade B, which has been reported to specifically label dead or dying neurons in the brain. Rats and mice were subjected to a moderate level of spinal cord injury using an IH impact device and sacrificed at 1, 2, 4, 7, 14, and 21 days post injury. Spinal cord tissue was processed for Fluoro-Jade B histochemistry and included sections throughout the injured region of the cord. No Fluoro-Jade positive neurons were observed in sections from any time point postinjury at any level of the spinal cord. Instead, Fluoro-Jade labeled astrocytes in uninjured control animals and injured animals. The specificity of astrocytic staining was confirmed by co-localizaton of Fluoro-Jade with glial fibrillary acidic protein. We also subjected a group of rats to a sequential cortical contusion injury and spinal cord injury. Sections from these animals showed numerous Fluoro-Jade positive neurons in the hippocampal formation and thalamus underlying the cortical contusion; however, the staining pattern in the spinal cord was identical to those animals that had received spinal cord injury alone.

Creatine diet supplement for spinal cord injury: influences on functional recovery and tissue sparing in rats.

Creatine-supplemented diet significantly attenuates cortical damage after traumatic brain injury in rodents. The protective mechanism likely involves maintenance of mitochondrial homeostasis. In the present study, we used two separate contusion spinal cord injury (SCI) instruments--the NYU device and the PSI Infinite Horizon (IH) impactor--to assess the efficacy of creatine-supplemented diets on hind limb functional recovery and tissue sparing in adult rats. Rats were fed control versus 2% creatine-supplemented chow for 4-5 weeks prior to SCI (pre-fed), after which most resumed a control diet while some remained on a 2% creatine diet (pre & post-fed). Following long-term behavioral analysis (BBB), the amount of spared spinal cord tissue among the dietary regimen groups was assessed using stereology. Comparatively, both instruments caused similar amounts of gray matter damage while the NYU device rendered a greater loss of white matter, reflected in more severe hind limb functional deficits than with the IH impactor. Relative to the control fed groups injured with either instrument, none of the creatine fed animals showed improvements in hind limb function or white matter tissue sparing. Although creatine did not attenuate gray matter loss in the NYU cohort, it significantly spared gray matter in the IH cohort with pre-fed and pre & post-fed regimens. Such selective sparing of injured spinal cord gray matter with a dietary supplement yields a promising strategy to promote neuroprotection after SCI. The relationship between the efficacy of creatine and the magnitude of the insults is discussed.

Experimental modeling of spinal cord injury: characterization of a force-defined injury device.

We examined the ability of a novel spinal cord injury (SCI) device to produce graded morphological and behavioral changes in the adult rat following an injury at thoracic level 10 (T10). The injury device uses force applied to the tissue as the control variable rather than tissue displacement. This has the advantage of eliminating errors that may arise from tissue movement prior to injury. Three different injury severities, defined by the amount of force applied to the exposed spinal cord at T10 (100, 150, and 200 kdyn), were evaluated at two different survival times (7 and 42 d). Unbiased stereology was employed to evaluate morphological differences following the injury. Quantitative behavioral assessment employed the Basso, Beattie, and Bresnahan locomotive rating scale. There was a significant force-related decline in locomotive ability following the injury. Animals subjected to a 200-kdyn injury performed significantly worse than animals subjected to a 100- and 150-kdyn injury. The locomotor ability at different days post injury significantly correlated with the amount of force applied to the spinal cord. Statistical analysis revealed several significant force-related morphological differences following the injury. The greatest loss of white and gray matter occurred at the site of injury impact and extended in both a rostral and caudal direction. Animals subjected to the greatest force (200 kdyn) displayed the least amount of spared tissue at both survival times indicative of the most severe injury. The amount of spared tissue significantly correlated with the locomotor ability. This novel rodent model of SCI provides a significant improvement over existing devices for SCI by reducing variability with a constant preset force to define the injury.

Efficacy of methylprednisolone therapy for the injured rat spinal cord.

Currently the synthetic glucocorticosteroid methylprednisolone sodium succinate (MPSS) is the standard therapy after acute spinal cord injury (SCI) in humans based on reported neurological improvements. The mechanisms for its beneficial actions are not entirely clear, but experimental evidence suggests MPSS affords some degree of neuroprotection. As many studies with rat models of SCI have been unable to demonstrate improved behavioral outcome or tissue sparing after MPSS treatment, we chose to stereologically assess whether it alters lesion volume and tissue sparing over time, as well as long-term behavioral recovery. Adult rats subjected to contusion SCI with the NYU impactor were administered either MPSS or saline for 24 hr beginning 5 min post injury. Over time the lesion dimensions were extremely dynamic, such that by 6 weeks post injury the volumes were reduced to a third of those seen after the first week. MPSS marginally reduced lesion volumes across time vs. controls, but the amount of spared gray and white matter remained unaltered between the two groups. Behavioral results further showed that MPSS failed to improve recovery of hind-limb function. These findings add to the emerging scrutiny of MPSS as the standard therapy for acute SCI, as well as indicate the existence of a therapeutic window for tissue sparing restricted to the first several days after this type of SCI in rats. Equally important, our results caution the use of lesion volume dimensions or percent tissue sparing at the epicenter as indicators of therapeutic efficacy because neither reflects the actual amount of tissue sparing.