Jun-ichiro Enmi

A Theoretical Model of Oxygen Delivery and Metabolism for Physiologic Interpretation of Quantitative Cerebral Blood Flow and Metabolic Rate of Oxygen

The coupling of cerebral blood flow (CBF) and metabolic rate of oxygen (CMRO2) during physiologic and pathophysiologic conditions remains the subject of debate. In the present study, we have developed a theoretical model for oxygen delivery and metabolism, which describes the diffusion of oxygen at the capillary-tissue interface and the nonlinear nature of hemoglobin (Hb) affinity to oxygen, allowing a variation in simple-capillary oxygen diffusibility, termed “effective oxygen diffusibility (EOD).” The model was used to simulate the relationship between CBF and CMRO2, as well as oxygen extraction fraction (OEF), when various pathophysiologic conditions were assumed involving functional activation, ischemia, hypoxia, anemia, or hypo- and hyper-capnic CBF variations. The simulations revealed that, to maintain CMRO2 constant, a variation in CBF and Hb required active change in EOD. In contrast, unless the EOD change took place, the brain allowed small but significant nonlinear change in CMRO2 directly dependent upon oxygen delivery. Application of the present model to quantitative neuroimaging of CBF and CMRO2 enables us to evaluate the biologic response at capillary level other than Hb- and flow-dependent properties of oxygen transport and may give us another insight regarding the physiologic control of oxygen delivery in the human brain.

A Novel Mouse Model of Subcortical Infarcts with Dementia

Subcortical white matter (WM) is a frequent target of ischemic injury and extensive WM lesions are important substrates of vascular cognitive impairment (VCI) in humans. However, ischemic stroke rodent models have been shown to mainly induce cerebral infarcts in the gray matter, while cerebral hypoperfusion models show only WM rarefaction without infarcts. The lack of animal models consistently replicating WM infarct damage may partially explain why many neuroprotective drugs for ischemic stroke or VCI have failed clinically, despite earlier success in preclinical experiments. Here, we report a novel animal model of WM infarct damage with cognitive impairment can be generated by surgical implantation of different devices to the right and left common carotid artery (CCA) in C57BL/6J mice. Implantation of an ameroid constrictor to the right CCA resulted in gradual occlusion of the vessel over 28 d, whereas placement of a microcoil to the left CCA induced ∼50% arterial stenosis. Arterial spin labeling showed a gradual reduction of cerebral blood flow over 28 d post operation. Such reductions were more marked in the right, compared with the left, hemisphere and in subcortical, rather than the cortical, areas. Histopathological analysis showed multiple infarct damage in right subcortical regions, including the corpus callosum, internal capsule, hippocampal fimbria, and caudoputamen, in 81% of mice. Mice displaying such damage performed significantly poorer in locomotor and cognitive tests. The current mouse model replicates the phenotypes of human subcortical VCI, including multiple WM infarcts with motor and cognitive impairment.

Experimental Pig Model of Old Myocardial Infarction with Long Survival Leading to Chronic Left Ventricular Dysfunction and Remodeling as Evaluated by PET

A pig model of reduced left ventricular (LV) function and remodeling or chronic heart failure with long survival after myocardial infarction (MI) has not been established. The aim of this study was to evaluate the pathophysiologic status of a pig model of old MI using a series of PET studies. Methods: Twenty-seven male farm pigs were divided into 2 groups: 7 animals in the control group and 20 animals that underwent a proximal coronary artery (CA) occlusion using an ameroid constrictor after distal CA ligation. A series of PET examinations was performed to assess LV volumes, LV functions, myocardial perfusion response to adenosine, and viability as water-perfusable tissue index. Results: The distal CA ligation inhibited arrhythmia during and after the operation, and a transmural anteroseptal MI, with an infarction area of 27% ± 5% of the whole left ventricle, was generated with a survival rate of 75% at 4 mo. Wall motion evaluated by 18F-FDG PET was diffusely reduced, including the noninfarcted wall. Global LV ejection fraction as assessed by gated C15O PET was reduced (39% ± 16%) in the group undergoing occlusion, compared with the control group (66% ± 16%, P < 0.05). LV end-systolic (31.4 ± 9.2 cm3) and end-diastolic (52.7 ± 10.2 cm3) volumes were increased, compared with controls (15.2 ± 9.4 cm3, P < 0.01, and 41.7 ± 11.5 cm3, P < 0.05, respectively). Histology showed hypertrophy and development of microscopic fibrosis in noninfarcted myocardium. PET demonstrated the reduced myocardial perfusion response to adenosine and also reduced water-perfusable tissue index in remote segments. Conclusion: The pig model of old MI generated by the chronic proximal CA obstruction after distal ligation was characterized by LV dysfunction and remodeling, with a high survival rate.

Design and characterization of a polymeric MRI contrast agent based on PVA for in vivo living‐cell tracking

A novel water-soluble MRI contrast agent for in vivo living cell tracking was developed. Unlike the conventional in vivo cell tracking system based on superparamagnetic iron oxide beads, the newly developed contrast agent is eliminated from the body when the contrast agent exits the cells upon cell death, which makes living cell tracking possible. The contrast agent is composed of gadolinium chelates (Gd-DOTA) and a water-soluble carrier, poly(vinyl alcohol) (PVA), which is known to interact with cells and tissues very weakly. Since the Gd-PVA was not taken up by cells spontaneously, the electroporation method was used for cell labeling. The delivered Gd-PVA was localized only in the cytosolic compartment of growing cells with low cytotoxicity and did not leak out of the living cells for long periods of time. This stability may be due to the weak cell-membrane affinity of Gd-PVA, and did not affect cell proliferation at all. After cell labeling, signal enhancement of cells was observed in vitro and in vivo. These results indicate that Gd-PVA can visualize only the living cells in vivo for a long period of time, even in areas deep within large animal bodies.

Gradual Carotid Artery Stenosis in Mice Closely Replicates Hypoperfusive Vascular Dementia in Humans.

Background Existing rodent models of vascular cognitive impairment (VCI) show abrupt changes in cerebral blood flow (CBF) and do not reliably replicate the clinical pathogenesis of VCI. We therefore aimed to develop a mouse model of VCI where CBF is gradually reduced, followed by subsequent progressive motor and cognitive impairment, after surgical intervention. Methods and Results Adult C57BL/6J male mice were subjected to gradual common carotid artery stenosis (GCAS) surgery by using an ameroid constrictor vessel‐constricting device with an inner diameter of 0.75 mm. The common carotid arteries narrowed gradually after gradual constriction of ameroid constrictors over 28 days after GCAS, with subsequent 79.3% area stenosis as a result of smooth muscle cell proliferation and macrophage infiltration in the tunica intima. The 28‐day survival rate was 91%. Arterial spin labeling demonstrated gradual and continuous reduction of cortical and subcortical CBF (ratio to the preoperative value) to 54.6% and 51.5%, respectively, over 28 days. However, magnetic resonance angiography showed increment of collateral flow signals in the leptomeningeal artery. Rarefaction and proliferation of astrocytes and microglia, with loss of oligodendrocytes, were found in the white matter at 32 days. Hippocampal neuronal loss was observed in only 25% of GCAS mice, consistent with lack of abnormalities in the Morris water maze test. The rotarod test showed motor impairment, and the Y‐maze test showed working memory deficits. Conclusions The GCAS model successfully generated gradual and continuous CBF reduction over 28 days, with replication of key histological, radiological, and behavioral features associated with cerebral hypoperfusion leading to VCI.

Validity of using a 3-dimensional PET scanner during inhalation of 15O-labeled oxygen for quantitative assessment of regional metabolic rate of oxygen in man

Use of 15O labeled oxygen (15O2) and positron emission tomography (PET) allows quantitative assessment of the regional metabolic rate of oxygen (CMRO2) in vivo, which is essential to understanding the pathological status of patients with cerebral vascular and neurological disorders. The method has, however, been challenging, when a 3D PET scanner is employed, largely attributed to the presence of gaseous radioactivity in the trachea and the inhalation system, which results in a large amount of scatter and random events in the PET assessment. The present study was intended to evaluate the adequacy of using a recently available commercial 3D PET scanner in the assessment of regional cerebral radioactivity distribution during an inhalation of 15O2. Systematic experiments were carried out on a brain phantom. Experiments were also performed on a healthy volunteer following a recently developed protocol for simultaneous assessment of CMRO2 and cerebral blood flow, which involves sequential administration of 15O2 and C15O2. A particular intention was to evaluate the adequacy of the scatter-correction procedures. The phantom experiment demonstrated that errors were within 3% at the practically maximum radioactivity in the face mask, with the greatest radioactivity in the lung. The volunteer experiment demonstrated that the counting rate was at peak during the 15O gas inhalation period, within a verified range. Tomographic images represented good quality over the entire FOV, including the lower part of the cerebral structures and the carotid artery regions. The scatter-correction procedures appeared to be important, particularly in the process to compensate for the scatter originating outside the FOV. Reconstructed images dramatically changed if the correction was carried out using inappropriate procedures. This study demonstrated that accurate reconstruction could be obtained when the scatter compensation was appropriately carried out. This study also suggested the feasibility of using a state-of-the-art 3D PET scanner in the quantitative PET imaging during inhalation of 15O labeled oxygen.

Long-Term/Bioinert Labeling of Rat Mesenchymal Stem Cells with PVA-Gd Conjugates and MRI Monitoring of the Labeled Cell Survival after Intramuscular Transplantation

Noninvasive in vivo imaging of transplanted stem cells is an effective method to clarify the mechanisms involved in stem cell transplantation therapy. We labeled rat mesenchymal stem cells (MSCs) with water-soluble magnetic resonance imaging (MRI) contrast agent poly(vinyl alcohol)-gadolinium (PVA-Gd) in order to ascertain the fate of transplanted MSCs in vivo. PVA-Gd was retained and localized in the cytosolic compartment of MSCs for a longer period of time. The effect of PVA-Gd labeling on MSC proliferation was much less than that of the commercially available contrast agent ProHance, and the labeled MSCs were found to have osteoblastic differentiation ability. To study the MSC lifetime in vivo, MSCs were seeded and trapped in the cytocompatible three-dimensional porous scaffolds of Spongel and transplanted. The MRI signal attributed to MSCs was eliminated from the transplanted site in 14 days. Because free PVA-Gd was rapidly eliminated from the site, this signal reduction indicated MSC death in the transplantation site. The low efficiency of MSC transplantation for ischemic tissue may be due to their short lifetime, making it important to develop highly effective stem cell transplantation systems that address cell number, injection position, and cell formulation (suspension, sheet, and aggregates). Our cell survival tracking system would be a very powerful tool to this end and would be applicable in clinical cell therapies.

Substantial Reduction of Parenchymal Cerebral Blood Flow in Mice with Bilateral Common Carotid Artery Stenosis

The bilateral common carotid artery stenosis (BCAS) mouse model, which replicates chronic cerebral hypoperfusion and white matter ischemic lesions, is considered to model some aspects of vascular cognitive impairment. Cerebral blood flow (CBF) changes in the brain surface post-BCAS have been demonstrated by laser speckle flowmetry, but CBF levels in the brain parenchyma remain unknown. Adult C57BL/6J male mice were subjected to BCAS using external microcoils. Brain magnetic resonance angiography (MRA) was conducted to visualize the intracranial main arteries while arterial spin labeling (ASL) was used to measure cortical and subcortical parenchymal CBF levels before and after BCAS. Brain MRA showed anterior circulation flow was substantially decreased until 14 days post-BCAS, which gradually but incompletely recovered over the following 14 days, with probable growth of collaterals from the posterior cerebral artery. ASL showed that cortical and subcortical parenchymal CBF remained decreased at approximately 50% of the baseline level during 1 and 14 days post-BCAS, recovering to approximately 70% at day 28. CBF levels in the parenchyma were lower than the cortical superficial region in the BCAS model and remained decreased without recovery during the first 2 weeks post-BCAS. These results suggest that the BCAS model reliably replicates chronic cerebral hypoperfusion.

Asymmetrical intersection between the middle cerebral artery and rhinal vein suggests asymmetrical gustatory cortex location in rodent hemispheres

The rodent gustatory cortex is located in the anterior part of the insular cortex, which is near the dorsal part of the rhinal vein (RHV) and the intersection of the anterior and posterior regions of the middle cerebral artery (MCA). Thus, the intersection between the RHV and MCA is used as a landmark for the rodent gustatory cortex. In our previous study, we employed functional magnetic resonance imaging (MRI) to demonstrate that tastants evoked bilateral responses in the rodent insular cortices, but that these representations were asymmetrical between the hemispheres. In the present study, to clarify the observed asymmetrical responses, we performed magnetic resonance angiography in a 7.0-Tesla MRI scanner to determine the anatomical position of the rodent gustatory cortex, which was identified using the intersection of the MCA and RHV. We successfully observed the intersection while administering carbogen as an inhaled gas and found that the intersection in the left hemisphere is more anterior compared to that in the right hemisphere. Taken together with the previous functional MRI results, this result indicates that the gustatory representation in relation to the intersection may be identically conserved in the insular cortex of both hemispheres; therefore, the rodent gustatory cortex may be asymmetrically located between the left and right hemispheres. The result also suggests that this landmark location needs to be verified when investigating gustatory representations and responses.

PET Quantification of Cerebral Oxygen Metabolism in Small Animals

Understanding cerebral oxygen metabolism is of great importance in both clinical diagnosis and animal experiments because oxygen is a fundamental source of brain energy and supports brain functional activities. Since small animals such as rats are widely used to study various diseases including cerebral ischemia, cerebrovascular diseases, and neurodegenerative diseases, the development of a noninvasive in vivo measurement method of cerebral oxygen metabolic parameters such as oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2) as well as cerebral blood flow (CBF) and cerebral blood volume (CBV) has been a priority. Although positron emission tomography (PET) with 15O labeled gas tracers has been recognized as a powerful way to evaluate cerebral oxygen metabolism in humans, this method could not be applied to rats due to technical problems and there were no reports of PET measurement of cerebral oxygen metabolism in rats until an 15O-O2 injection method was developed a decade ago. Herein, we introduce an intravenous administration method using two types of injectable 15O-O2 and an 15O-O2 gas inhalation method through an airway placed in the trachea, which enables oxygen metabolism measurements in rats.