Background and Purpose-The present studies examined the hypothesis that the distribution of cerebral injury after a focal ischemic insult is associated with the regional distribution of nitric oxide synthase (NOS) activity. Methods-Based on previous studies that certain anatomically well-defined areas are prone to become either core or penumbra after middle cerebral artery occlusion (MCAO), we measured NOS activity in these areas from the right and left hemispheres in a spontaneously hypertensive rat filament model. Four groups were studied: (1) controls (immediate decapitation); (2) 1.5 hours of MCAO with no reperfusion (R0); (3) 1.5 hours of MCAO with 0.5 hour of reperfusion (R0.5); and (4) 1.5 hours of MCAO with 24 hours of reperfusion (R24). Three groups of corresponding isoflurane sham controls were also included: 1.5 (S1.5) or 2 (S2.0) hours of anesthesia and 1.5 hours of anesthesiaϩ24 hours of observation (S24). Results-Control core NOS activity for combined right and left hemispheres was 129% greater than penumbral NOS activity (PϽ0.05). Combined core NOS activity was also greater (PϽ0.05) in the three sham groups: 208%, 122%, and 161%, respectively. In the three MCAO groups, ischemic and nonischemic core NOS remained higher than penumbral regions (PϽ0.05). However, NOS activity was lower in the ischemic than in the nonischemic core in all three groups: R0 (29% lower), R0.5 (48%), and R24 (86%) (PϽ0.05). Addition of cofactors (10 mol/L tetrahydrobiopterin, 3 mol/L flavin adenine dinucleotide, and 3 mol/L flavin mononucleotide) increased NOS activity in all groups and lessened the decrease in ischemic core and penumbral NOS. Conclusions-Greater NOS activity in core regions could explain in part the increased vulnerability of that region to ischemia and could theoretically contribute to the progression of the infarct over time. The data also suggest that NOS activity during ischemia and reperfusion could be influenced by the availability of cofactors.

Using an 11.7-Tesla magnetic resonance imaging (MRI) scanner in 10-d-old rat pups we report on the evolution of injury over 28 d in a model of neonatal stroke (transient filament middle cerebral artery occlusion, tfMCAO) and a model of hypoxicischemic injury (Rice-Vannucci model, RVM). In both models, diffusion-weighted imaging (DWI) was more sensitive in the early detection of ischemia than T2-weighted imaging (T2WI). Injury volumes in both models were greater on d 1 for DWI and d 3 for T2WI, decreased over time and by d 28 T2WI injury volumes (tfMCAO 10.3% of ipsilateral hemisphere; RVM 23.9%) were definable. The distribution of injury with tfMCAO was confined to the vascular territory of the middle cerebral artery and a definable core and penumbra evolved over time. Ischemic injury in the RVM was more generalized and greater in cortical regions. Contralateral hemispheric involvement was only observed in the RVM. Our findings demonstrate that high-field MRI over extended periods of time is possible in a small animal model of neonatal brain injury and that the tfMCAO model should be used for studies of neonatal stroke and that the RVM does not reflect the vascular distribution of injury seen with focal ischemia. (Pediatr Res 61: 9-14, 2007) C urrent neuroprotective therapies of acute stroke in adults have not proven beneficial and because the delay in diagnosis of neonatal stroke is beyond the current window of opportunity for thrombolytic therapy, it is likely that future neonatal care will focus on delayed treatments implemented hours to days after ischemia. Temporally delayed treatment is even more likely in cases of neonatal ischemia as observable signs are more difficult to assess and observe. As such, being able to serially and noninvasively monitor the evolution of neonatal stroke in humans as well as animal models over weeks to months would be advantageous in studying mechanisms of injury, repair and responses to treatment. Magnetic resonance imaging (MRI) has greatly improved our ability to detect stroke in newborns but its use in neonatal stroke models has been limited by the technical challenges in serially acquiring such data (1,2).The current study used high field strength (11.7T; Tesla) MRI to study the evolution of neonatal hypoxic-ischemic injury over a period of 28 d in two different models. We chose to compare a model of neonatal stroke (tfMCAO, transient filament middle cerebral artery occlusion) to that of the more commonly used model of neonatal hypoxic-ischemic brain injury (RVM, Rice-Vannucci model of unilateral carotid ligation and 1.5 h of hypoxia). We hypothesized that MRI could be used in a small animal model to serially and noninvasively monitor the evolution of injury over 28 d and that neuroimaging could elucidate temporal and spatial patterns of injury in the two models. Specifically, we wished to determine whether there were neuroimaging differences between the two models in: (1) the distribution of injury; (2) the degree of injury in striatum and cortex; (3) the evolut...

We report a new clinically relevant model of neonatal hypoxic-ischemic injury in a 10-day-old rat pup. Bilateral carotid artery occlusion and 8% hypoxia (1 to 15 mins, BCAO-H) was induced with varying degrees of injury (mild, moderate, severe), which was quantified using magnetic resonance imaging including diffusion-weighted and T2-weighted imaging at 24 h and 21/28 days. We developed a magnetic resonance imaging-based rat pup severity score and compared 3D ischemic injury volumes/rat pup severity score with histology and behavioral testing. At 24 h, hypoxicischemic injury was observed in 17/27 animals; long-term survival was 81%. Magnetic resonance imaging lesion volumes did not correlate with hypoxia duration but correlated with rat pup severity score, which was used to classify animals into mild (n = 21), moderate (n = 6), and severe (n = 10) groups with average brain lesion volumes of 0.9%, 33.2%, and 56.3%, respectively. Histology confirmed lesion location and histologic scoring correlated with the rat pup severity score. We also found excellent correlation between injury severity and multiple behavioral tasks. Bilateral carotid artery occlusion and hypoxia in the P10 rat pup is an excellent model of neonatal hypoxic-ischemic injury because it induces diffuse global injury similar to the term infant. This model can produce graded injury severity, similar to that seen in human neonates, but manipulation with hypoxia duration is unpredictable.

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