Beatriz Tone

Core and Penumbral Nitric Oxide Synthase Activity During Cerebral Ischemia and Reperfusion

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 anesthesia+24 hours of observation (S24). RESULTS Control core NOS activity for combined right and left hemispheres was 129% greater than penumbral NOS activity (P<0.05). Combined core NOS activity was also greater (P<0.05) 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 (P<0.05). 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%) (P<0.05). Addition of cofactors (10 micromol/L tetrahydrobiopterin, 3 micromol/L flavin adenine dinucleotide, and 3 micromol/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.

Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference

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.

Temporal and regional evolution of aquaporin-4 expression and magnetic resonance imaging in a rat pup model of neonatal stroke.

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.

Long‐term magnetic resonance imaging of stem cells in neonatal ischemic injury

Quantitative magnetic resonance imaging (MRI) can serially and noninvasively assess the degree of injury in rat pup models of hypoxic ischemic injury (HII). It can also noninvasively monitor stem cell migration following iron oxide prelabeling. 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 noninvasively 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.

Comparison of Two Neonatal Ischemic Injury Models Using Magnetic Resonance Imaging

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 hypoxic-ischemic 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.

Automated core-penumbra quantification in neonatal ischemic brain injury.

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 diffusion-perfusion 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.

Rodent Neonatal Bilateral Carotid Artery Occlusion with Hypoxia Mimics Human Hypoxic-Ischemic Injury

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, hypoxic-ischemic 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.

Albumin reduces blood-brain barrier permeability but does not alter infarct size in a rat model of neonatal stroke.

Human serum albumin therapy confers neurobehavioral and histopathologic neuroprotection in adult stroke models. We investigated whether albumin might also be neuroprotective in ischemic brain injury using a transient filament middle cerebral artery occlusion (tfMCAO) model in 10-d-old rat pups treated with 0.25% albumin or saline 1 h after reperfusion. We performed serial neurobehavioral and magnetic resonance imaging (MRI) assessments immediately after tfMCAO (day 0) and on 1, 3, 7, 14, and 28 d. IgG staining to assess blood-brain barrier (BBB) integrity and standard histology was obtained on 1, 3, and 28 d. Hemispheric infarct volumes from MRI were similar in saline and albumin groups (0 h: 39% and 44%; d 1: 46% and 55%; and d 28:10% and 24%) as were neurobehavioral assessments. IgG staining at 3 d post-ischemia showed loss of BBB integrity that was significantly reduced after albumin. Elevated T2 values suggesting vasogenic edema was seen in albumin compared with saline-treated animals, as was increased water mobility (i.e. increased apparent diffusion coefficient (ADC) reflecting cytotoxic edema. The reasons why albumin was not neuroprotective in neonatal stroke compared with adults remain uncertain. Effective strategies in adult models need to be reassessed in the neonate.

Serial magnetic resonance imaging in a rat pup filament stroke model.

Neonatal stroke is increasingly recognized in preterm and term infants but the ability to study this condition has been limited by the technical challenges in developing suitable animal models. In the current study we report the use of transient filament middle cerebral artery occlusion for 1.5 h in 10-day-old rat pups in which we were able to perform serial magnetic resonance imaging (MRI) studies. Serial MRI was performed immediately after the onset of stroke until 28 days after injury in an 11.7 T scanner using diffusion weighted and T2-weighted images. At 28 days the rat pups were sacrificed and standard histological stains were performed to validate stroke area. Serial behavioral assessments were also performed on the day of each imaging study. The anatomical distribution of stroke was similar to that expected from occlusion of the middle cerebral artery in adult models and represents a specific model of neonatal stroke in contrast to the commonly used model of carotid artery occlusion with 8% hypoxia. The initial stroke volume from MR measurements was 39% of the ipsilateral hemisphere at 0 h post-occlusion, reached a maximum at 24 h (44%) and then decreased in size (17%) with subsequent cavitation by 28 days. Infarction was more visible early with diffusion weighted imaging whereas T2-mapping provided more accurate infarct volumes at later time points. Despite the relatively large infarct volume, we saw little evidence of behavioral neurological deficit suggesting that this may also serve as a model of developmental plasticity and recovery.

Core and penumbral nitric oxide synthase activity during cerebral ischemia and reperfusion in the rat pup

Our studies examined the hypothesis that the distribution of cerebral injury after a focal ischemic insult in the immature rat pup is associated with the regional distribution of nitric oxide synthase (NOS) activity and that differences in the vulnerability to ischemia between pup and adult might be related to differences in cofactor availability. We measured NOS activity in well-defined regions prone to become either core or penumbra in controls and at different times (end of occlusion, 0.5 h, and 24 h reperfusion) after middle cerebral artery occlusion (MCAO) from the right and left hemispheres in a 14- to 18-day-old rat pup filament model. Three groups of corresponding isoflurane sham controls were also included. “Core” NOS activity for combined right and left hemispheres ranged from 113% to 217% more than “penumbral” regions in control and sham groups. In the three MCAO groups, marked decreases in ischemic core and penumbral NOS activity were seen; however, core NOS remained higher than penumbral regions bilaterally. The effects of cofactor addition (10 μM tetrahydrobiopterin, 3 μM flavin adenine dinucleotide, and 3 μM flavin mononucleotide) on NOS activity were similar in “core” and “penumbral” regions in control and sham groups. However, after 24 h MCAO, cofactor addition preferentially increased NOS activity in the ischemic hemisphere. Cofactor addition in the pup also had a greater effect on enhancing NOS activity in all regions compared with the adult. Greater NOS activity in core regions in the rat pup, as in the adult, could in part, explain the increased vulnerability of that region to ischemia. NOS activity also can be influenced by the availability of cofactors and this effect may be greater in the immature animal.