A comparison of two biomaterial carriers for osteogenic protein-1 (BMP-7) in an ovine critical defect model.

Critical size defects in ovine tibiae, stabilised with intramedullary interlocking nails, were used to assess whether the addition of carboxymethylcellulose to the standard osteogenic protein-1 (OP-1/BMP-7) implant would affect the implant's efficacy for bone regeneration. The biomaterial carriers were a 'putty' carrier of carboxymethylcellulose and bovine-derived type-I collagen (OPP) or the standard with collagen alone (OPC). These two treatments were also compared to "ungrafted" negative controls. Efficacy of regeneration was determined using radiological, biomechanical and histological evaluations after four months of healing. The defects, filled with OPP and OPC, demonstrated radiodense material spanning the defect after one month of healing, with radiographic evidence of recorticalisation and remodelling by two months. The OPP and OPC treatment groups had equivalent structural and material properties that were significantly greater than those in the ungrafted controls. The structural properties of the OPP- and OPC-treated limbs were equivalent to those of the contralateral untreated limb (p > 0.05), yet material properties were inferior (p < 0.05). Histopathology revealed no residual inflammatory response to the biomaterial carriers or OP-1. The OPP- and OPC-treated animals had 60% to 85% lamellar bone within the defect, and less than 25% of the regenerate was composed of fibrous tissue. The defects in the untreated control animals contained less than 40% lamellar bone and more than 60% was fibrous tissue, creating full cortical thickness defects. In our studies carboxymethylcellulose did not adversely affect the capacity of the standard OP-1 implant for regenerating bone.

Secondary elevation of extracellular neurotransmitter amino acids in the reperfusion phase following focal cerebral ischemia.

The purpose of this study was to evaluate amino acid neurotransmitter dynamics in the reperfusion phase after transient cerebral ischemia. In vivo microdialysis was used to measure extracellular amino acid levels in a rabbit model of focal ischemia. During 30 min of transient ischemia (n = 5), small but significant (p < 0.05) increases in glutamate, aspartate, gamma-aminobutyric acid (GABA), and taurine were noted. These elevations rapidly returned to baseline levels upon recirculation and remained constant for up to 5.5 h of reperfusion. In rabbits subjected to 2 h of transient ischemia (n = 5), two phases of amino acid release were seen. During ischemia, large (5- to 50-fold) elevations in glutamate, aspartate, GABA, and taurine occurred, as expected. These elevations rapidly normalized upon unocclusion. However, significant (p < 0.05) secondary elevations in glutamate, aspartate, and GABA occurred after 2-4 h of reperfusion. Regression analysis demonstrated significant correlations between primary (ischemic) and secondary (reperfusion) efflux. In permanent ischemia (n = 5), amino acid levels remained elevated throughout the entire experiment. Secondary elevations in excitatory amino acids may further contribute to the excitotoxic cascade during reperfusion.

Pharmacologic reversal of acute changes in diffusion-weighted magnetic resonance imaging in focal cerebral ischemia.

Recently, diffusion-weighted magnetic resonance imaging (DWI) has been shown to visualize acute ischemic lesions in the brain before changes are observable with conventional magnetic resonance imaging. However, the underlying mechanisms of these acute DWI changes are unclear and may include both reversible and irreversible damage. In this study, we demonstrate that acute DWI lesions may be reversed with MK801 therapy postischemia. Sprague-Dawley rats (n = 12) were subjected to middle cerebral artery occlusion and DWI scans were obtained beginning 60 min postocclusion. Distinct regions of hyperintensity were observed in the basal ganglia and cortex, corresponding with the expected distribution of ischemia in this model. After the first scan, animals were treated with MK801 (0.5 mg/kg i.v.) or normal saline and subsequently scanned again 30 and 60 min after treatment. In the control group, the area of hyperintense lesions continued to increase, by 55% in the cortex and 57% in the basal ganglia. MK801 therapy significantly (p < 0.01) reduced the area of damage by the third DWI scan at 60 min posttreatment (-50% cortex, -22% basal ganglia, -41% total hemisphere) compared to pretreatment scans. Tetrazolium (TTC) stains at 24 h confirmed that MK801 significantly reduced the volumes of infarction (p < 0.05). These results demonstrate that significant portions of the acute ischemic lesion on DWI are reversible with pharmacologic intervention.

MRI measurements of water diffusion and cerebral perfusion: their relationship in a rat model of focal cerebral ischemia.

The aim of this study was to examine the quantitative relationship between changes in apparent diffusion coefficient (ADC) and transverse relaxivity (delta R2*) measurements of relative perfusion deficits within the gradients of a focal ischemic insult. Sixty minutes after permanent occlusion of the middle cerebral artery, rats (n = 7) were subjected to spin echo diffusion-weighted scans followed by fast low-angle shot (FLASH) perfusion-sensitive scans. Diffusion-weighted images showed clear ischemic lesions in the affected basal ganglia and cortex. Ischemic deficits were demonstrated as a decrease in first-pass transit of injected boluses of gadodiamide. ADC maps were generated and regions of interest (ROIs) were obtained to span the range of ADC reductions from the lesion center or core to the periphery or penumbra. Corresponding ROIs from the bolus injection images were used to calculate perfusion indexes relative to contralateral levels as ratios of delta R2* integrals and ratios of delta R2* peak values. In all animals, the degree of ADC reductions was related to the degree of delta R2* perfusion deficits, ranging from severe ischemia in the core of the lesion to intermediate and moderate changes toward the lesion periphery. In the ischemic periphery, ADC reductions were linearly correlated with delta R2* peak ratios. However, no significant correlation was found between ADC reductions and delta R2* integral ratios. These data suggest that magnetic resonance measurements of ADC and delta R2* peak ratios can be used to quantitatively assess the variable gradients in focal ischemia, including potentiallyn critical areas at risk in the ischemic periphery.

Alterations in K+ evoked profiles of neurotransmitter and neuromodulator amino acids after focal ischemia-reperfusion.

Secondary elevations in extracellular amino acids occur during reperfusion after transient cerebral ischemia. The delayed accumulation of excitatory amino acids may contribute to the progressive development of neuronal injury. In this study, we explored the mechanisms that may be involved in this phenomenon. Microdialysis samples from probes located in rabbit cortex were analysed with a chiral amino acid procedure. Concentrations of neurotransmitters (L-Glu, GABA), N-methyl-D-aspartate receptor modulators (D-Ser, Gly), an inhibitory neuromodulator (Tau), the lipid component phosphoethanolamine, and L-Gln, L-Ser and L-Ala were measured. Depolarization via perfusion with potassium was used to assess the status of release/reuptake systems at 2 and 4 h reperfusion after 2 h transient focal ischemia. Background experiments classified potassium evoked responses as calcium dependent or calcium-independent by inclusion of 30 microM omega-conopeptide MVIIC or by inclusion of 20 mM magnesium and ommision of calcium. During ischemia, large elevations of almost all amino acids occurred. During reperfusion, secondary elevations in transmitter amino acids (L-Glu, GABA) and N-methyl-D-aspartate receptor modulators (D-Ser, Gly) occurred. Tau remained slightly elevated whereas the lipid component phosphoethanolamine remained high and stable during reperfusion. Reperfusion significantly potentiated the potassium response for amino acids with calcium-dependent responses (L-Glu and GABA). In contrast, calcium-independent responses (Tau, phosphoethanolamine, L-Gln) were significantly attenuated. Intermediate behavior was observed with Gly, while no potassium responses were observed for D-Ser, L-Ser or L-Ala. These data demonstrate that perturbations in evoked amino acid profiles after ischemia-reperfusion are selective. Reduction of calcium-independent responses implicate a general decline in efficacy of transporter mechanisms that restore transmembrane gradients of ions and transmitters. Decreased efficacy of transporter systems may reduce transmitter reuptake and account for the amplified release of L-Glu and GABA, thus contributing to progressive neural dysfunction after cerebral ischemia.

Characterization of mitochondrial glutaminase and amino acids at prolonged times after experimental focal cerebral ischemia.

The mitochondrial enzyme glutaminase is a significant contributor to extracellular glutamate after neuronal injury in vitro [R. Newcomb, X. Sun, L. Taylor, N. Curthoys, R.G. Giffard, Increased production of extracellular glutamate by the mitochondrial glutaminase following neuronal death, J. Biol. Chem. 272 (1997) 11276-11282.]. As a step towards characterizing the role of the enzyme in neuronal injury in vivo, glutaminase activity was measured in central and peripheral regions of the ischemic distribution in rat brain at 6, 24, and 48 h after permanent focal ischemia. Although glutaminase activity decreases in the central ischemic area, significant activity remains in peripheral areas of evolving damage, even after 24 and 48 h ischemia. Western blots show no detectable change in glutaminase molecular weight or total immunoreactivity, regardless of the degree of inactivation. Significant amounts of glutamine remain in ischemic tissue at prolonged times after focal ischemia, while reductions in tissue amounts of glutamate are highly correlated with decreases in glutaminase activity. In vivo microdialysis probes were inserted into the ischemic periphery after 24 h focal ischemia. Glutamate is significantly elevated in these dialysates. Perfusion of the glutaminase substrate glutamine and the enzyme activator phosphate results in further and specific elevations in dialysate glutamate. In sum, significant mitochondrial glutaminase activity remains in the periphery of the ischemic lesion at 24 and 48 h, where it can contribute directly to elevated extracellular glutamate. Inactivation of the glutaminase in central areas of the ischemic lesion does not involve significant proteolytic degradation, and likely involves a specific molecular event.

Role of vasogenic edema and tissue cavitation in ischemic evolution on diffusion-weighted imaging: comparison with multiparameter MR and immunohistochemistry.

PURPOSE: To examine the mechanisms of further evolution that occurs from the early to late phase after initial changes in diffusion-weighted imaging after cerebral ischemia. METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion. Diffusion-, proton density-, T1- and T2-weighted imaging were performed on days 0, 2, and 6. Histologic examination (IgG, glial fibrillary acidic protein, and cresyl violet staining) was done after scanning. RESULTS: Apparent diffusion coefficients (ADCs) in the ischemic hemisphere were significantly decreased on day 0. Thereafter, ADCs increased over time and became significantly higher than the contralateral side by day 6. Changes in basal ganglia occurred more rapidly than in cortex. Proton density-, T1-, and T2-weighted scans showed maximal changes on day 2. From day 0 to day 2, there are significant correlations between changes in ADC and changes in T1-weighted signals and T2-weighted signals. Histologic exam showed early neuronal injury on day 0, intense gliotic activity and protein leakage associated with infarction and edema on day 2, and cavitation in severely infarcted areas on day 6. CONCLUSION: After initial reduction of ADC, the subsequent increase in ADC values on day 2 may be associated with vasogenic edema and cell lysis. Later elevations in ADC may be related to cavitation of infarcted tissue.

Neuroprotection with NBQX in rat focal cerebral ischemia. Effects on ADC probability distribution functions and diffusion-perfusion relationships.

BACKGROUND AND PURPOSE: We have previously shown that treatment with glutamate receptor antagonists after focal ischemia can partially reverse acute lesions measured with diffusion-weighted MRI. The goal of this study was to examine the quantitative nature of these effects of neuroprotection. METHODS: Rats were subjected to permanent occlusion of the middle cerebral artery under halothane anesthesia and treated with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) (30 mg/kg IP; two doses given immediately after ischemia and 1 hour after ischemia) or given injections of saline. Diffusion-weighted MRI scans were performed to map the changes in water diffusivity during the first 3 hours after ischemia. Apparent diffusion coefficients (ADCs) within the ischemic hemisphere were calculated, and ischemic changes were expressed as absolute reductions and as a percentage of contralateral mean values. Relative perfusion deficits in the ischemic hemisphere were assessed with dynamic MRI of transient changes in transverse relaxation rates (delta R2*). RESULTS: Analysis with ADC probability distribution functions showed that focal ischemia was present with gradients in ADC reductions emanating from the center to the periphery of the lesion. Ischemic evolution in control rats was manifested as a progressive shift of the probability distribution functions over time. NBQX treatment resulted in a reverse shift of these probability functions. By 3 hours after occlusion, probability distribution functions were significantly improved in treated rats (P < .05). Because of the temporal evolution of the probability distribution functions, ADC thresholds that correlated with histological outcomes of infarction changed over time. NBQX did not alter the cerebral perfusion index, measured as delta R2* peak values. CONCLUSIONS: The results indicate that ADC probability distribution functions can be used to quantitatively evaluate the effects of neuroprotective treatment on the gradients of injury in focal cerebral ischemia. The probability functions also allow for intrasubject comparisons and may therefore be useful for exploring therapeutic windows.

Temporal correlation mapping analysis of the hemodynamic penumbra in mutant mice deficient in endothelial nitric oxide synthase gene expression.

BACKGROUND AND PURPOSE: Mice containing deletions in the genes encoding nitric oxide (NO) synthase have been useful to dissect the role of NO in cerebral ischemia. We recently reported that mice lacking expression of the endothelial isoform of NO synthase (eNOS) develop larger infarcts after middle cerebral artery occlusion. Because NO or a related product of NO synthase activity is important for relaxation of cerebral blood vessels, we examined for possible hemodynamic differences in the peri-ischemic zone of eNOS-deficient and wild-type mice after middle cerebral artery occlusion using functional CT scanning techniques. METHODS: Wild-type SV129 mice (n = 10) and mice deficient in eNOS gene expression (n = 10) were subjected to middle cerebral artery occlusion under halothane anesthesia. Thirty minutes after ischemia, functional CT scanning was performed with dynamic scanning protocols to measure the cerebral transit profiles of injected contrast agents. A temporal correlation mapping technique was used to analyze the pattern of hemodynamic perturbations based on alterations in the shape of the cerebral transit profiles. Statistical thresholds defined the hemodynamic core and penumbra. RESULTS: Hemodynamic deficits were more severe in the mutant than wild-type mouse. When expressed as a percentage of the total insult, core areas were significantly increased in mutant mice (39.8 +/- 3.7%) compared with wild types (28.8 +/- 3.4%). Conversely, areas of the hemodynamic penumbra were significantly smaller in mice deficient in eNOS activity (60.2 +/- 3.7%) than in wild-type mice (71.2 +/- 3.4%). Furthermore, the calculated relative perfusion index within the hemodynamic penumbra was significantly lower in the group with eNOS gene deletion (35.6 +/- 1.5% in mutants versus 43.0 +/- 2.4% in wild types). CONCLUSIONS: These data indicate that mice lacking eNOS expression show a greater degree of hemodynamic compromise after middle cerebral artery occlusion and suggest that a product of eNOS activity (eg. NO) may protect brain after focal cerebral ischemia, possibly by improving blood flow within the penumbral zone.