Neuroimaging with diffusion-weighted imaging is routinely used for clinical diagnosis/prognosis. Its quantitative parameter, the apparent diffusion coefficient (ADC), is thought to reflect water mobility in brain tissues. After injury, reduced ADC values are thought to be secondary to decreases in the extracellular space caused by cell swelling. However, the physiological mechanisms associated with such changes remain uncertain. Aquaporins (AQPs) facilitate water diffusion through the plasma membrane and provide a unique opportunity to examine the molecular mechanisms underlying water mobility. Because of this critical role and the recognition that brain AQP4 is distributed within astrocytic cell membranes, we hypothesized that AQP4 contributes to the regulation of water diffusion and variations in its expression would alter ADC values in normal brain. Using RNA interference in the rodent brain, we acutely knocked down AQP4 expression and observed that a 27% AQP4-specific silencing induced a 50% decrease in ADC values, without modification of tissue histology. Our results demonstrate that ADC values in normal brain are modulated by astrocytic AQP4. These findings have major clinical relevance as they suggest that imaging changes seen in acute neurologic disorders such as stroke and trauma are in part due to changes in tissue AQP4 levels.

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.

Objective Quantitative magnetic resonance imaging (MRI) can serially and non-invasively assess the degree of injury in rat pup models of hypoxic ischemic injury (HII). It can also non-invasively monitor stem cell migration following iron oxide pre-labeling. Reports have shown that neural stem cells (NSCs) may help mediate neuroprotection or stimulate neuroreparative responses in adult and neonatal models of ischemic injury. We investigated the ability of high-field MRI to monitor and non-invasively quantify the migration, proliferation, and location of iron oxide-labeled NSCs over very long time periods (58 weeks) in real time while contemporaneously correlating this activity with the evolving severity and extent of neural damage. Methods Labeled clonal murine NSCs were implanted 3 days after unilateral HII in 10 day old rat pups into the contralateral striatum or ventricle. We developed methods for objectively quantifying key aspects of dynamic NSC behavior (e.g., viability; extent and speed of migration; degree of proliferation; extent of integration into host parenchyma). MRI images were validated with histological and immunohistochemical assessments. Results mNSCs rapidly migrated (100μm/day) to the lesion site. Chains of migrating NSCs were observed in the corpus callosum. In pups subjected to HII, though not in intact control animals, we observed a 273% increase in the MR-derived volume of mNSCs 4 weeks after implantation (correlating with the known proliferative behavior of endogenous and exogenous NSCs) that slowly declined over the 58 week time course, with no adverse consequences. Large numbers of now quiescent mNSCs remained at the site of injury, many retaining their iron oxide label. Interpretation Our studies demonstrate that MRI can simultaneously monitor evolving neonatal cerebral injury as well as NSC migration and location. Most importantly, it can non-invasively monitor proliferation dynamically for prolonged time periods. To be able to pursue clinical trials in newborns using stem cell therapies, it is axiomatic that safety be insured through the long-term real time monitoring of cell fate and activity, particularly with regard to observing unanticipated risks to the developing brain. This study supports the feasibility of reliably using MRI for this purpose.

Edema formation can be observed using magnetic resonance imaging (MRI) in patients with stroke. Recent studies have shown that aquaporin-4 (AQP4), a water channel, is induced early after stroke and potentially participates in the development of brain edema. We studied whether induction of AQP4 correlated with edema formation in a rat pup filament stroke model using high field (11.7-Tesla) MRI followed by immunohistochemical investigation of AQP4 protein expression. At 24 h, we observed increased T2 values and decreased apparent diffusion coefficients (ADC) within injured cortical and striatal regions that reflected the edema formation. Coincident with these MR changes were significant increases in AQP4 expression on astrocytic end-feet in the border regions of injured tissues. Striatal imaging findings were still present at 72 h with a slow normalization of AQP4 expression in the border regions. At 28 d, AQP4 expression normalized in the border while in this region ADC values increased. We show that induction of AQP4 is increased during the period of active edema formation in the border region without regional correlation with edema. Finally, induction of AQP4 on astrocyte end-feet could participate in tissue preservation after ischemia in the immature rat brain. A rterial ischemic stroke, often within the distribution of the MCA, occurs in approximately 1 per 4,000 neonates and as in adults there are significant sequelae over the lifespan that make this disease an important public health concern (1). Increasingly, magnetic resonance imaging (MRI) is used to evaluate neonatal ischemic brain injury and several recent studies have demonstrated the added value of DWI in assessing injury severity and in determining long-term outcome (2-4). MRI also has been used to visualize brain edema that contributes to the morbidity and mortality of many of these conditions (5). In general, diffusion restriction with reduced ADC values correlates with cytotoxic edema, whereas increased T2WI values reflects the development of vasogenic edema (6,7).In the past decade, attention has focused on AQP in the development of brain edema. AQP are water-specific channels that provide a direct water route through the plasma membrane and are important in the regulation of water movement. Expression of AQP4 and AQP9, detected in mammalian brain, is altered in several conditions, including ischemia (7-9). Although astrocytic expression of AQP4 and AQP9 increases after ischemia, only AQP4 expression temporally correlates with the evolution of brain edema in adult mice and suggests that regulation of AQP4 expression could be a potential therapeutic target (9).The role of AQP4 in the evolution of cytotoxic and vasogenic edema after stroke remains uncertain. In the adult mouse, increased astrocytic AQP4 expression was observed at 1 h and 48 h after stroke (9). In contrast to adult stroke model studies, investigations in a neonatal rat pup model of HII found that AQP4 expression (decreased at 1 h and 24 h after HII), was associated with d...

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...

Neonatal hypoxic-ischemic brain injury (HII) and arterial ischemic stroke (AIS) result in irreversibly injured (core) and salvageable (penumbral) tissue regions. Identification and reliable quantification of salvageable tissue is pivotal to any effective and safe intervention. Magnetic resonance imaging (MRI) is the current standard to distinguish core from penumbra using diffusionperfusion mismatch (DPM). However, subtle MR signal variations between core-penumbral regions make their visual delineation difficult. We hypothesized that computational analysis of MRI data provides a more accurate assessment of core and penumbral tissue evolution in HII/AIS. We used two neonatal rat-pup models of HII/AIS (unilateral and global hypoxic-ischemia) and clinical data sets from neonates with AIS to test our noninvasive, automated computational approach, Hierarchical Region Splitting (HRS), to detect and quantify ischemic core-penumbra using only a single MRI modality (T2-or diffusion-weighted imaging, T2WI/DWI). We also validated our approach by comparing core-penumbral images (from HRS) to DPM with immunohistochemical validation of HII tissues. Our translational and clinical data results showed that HRS could accurately and reliably distinguish the ischemic core from penumbra and their spatiotemporal evolution, which may aid in the vetting and execution of effective therapeutic interventions as well as patient selection.

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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