Acetyl-CoA carboxylase 2 inhibition reduces skeletal muscle bioactive lipid content and attenuates progression of type 2 diabetes in Zucker diabetic fatty rats.

Intramyocellular lipid (IMCL) accumulation in skeletal muscle is closely associated with development of insulin resistance. In particular, diacylglycerol and ceramide are currently considered as causal bioactive lipids for impaired insulin action. Recently, inhibition of acetyl-CoA carboxylase 2 (ACC2), which negatively modulates mitochondrial fatty acid oxidation, has been shown to reduce total IMCL content and improve whole-body insulin resistance. This study aimed to investigate whether ACC2 inhibition-induced compositional changes in bioactive lipids, especially diacylglycerol and ceramide, within skeletal muscle contribute to the improved insulin resistance. In skeletal muscle of normal rats, treatment of the ACC2 inhibitor compound 2e significantly decreased both diacylglycerol and ceramide levels while having no significant impact on other lipid metabolite levels. In skeletal muscle of Zucker diabetic fatty (ZDF) rats, which exhibited greater lipid accumulation than that of normal rats, compound 2e significantly decreased diacylglycerol and ceramide levels corresponding to reduced long chain acyl-CoA pools. Additionally, in the lipid metabolomics study, ZDF rats treated with compound 2e also showed improved diabetes-related metabolic disturbance, as reflected by delayed hyperinsulinemia as well as upregulated gene expression associated with diabetic conditions in skeletal muscle. These metabolic improvements were strongly correlated with the bioactive lipid reductions. Furthermore, long-term treatment of compound 2e markedly improved whole-body insulin resistance, attenuated hyperglycemia and delayed insulin secretion defect even at severe diabetic conditions. These findings suggest that ACC2 inhibition decreases diacylglycerol and ceramide accumulation within skeletal muscle by enhancing acyl-CoA breakdown, leading to attenuation of lipid-induced insulin resistance and subsequent diabetes progression.

Pharmacological MRI responses of raclopride in rats: The relationship with D2 receptor occupancy and cataleptic behavior.

Pharmacological magnetic resonance imaging (phMRI) allows the visualization of brain pharmacological effects of drugs using functional MRI (fMRI). phMRI can help us facilitate central nervous system (CNS) drug development. However, there have been few studies demonstrating the dose relationship of the fMRI response induced by CNS drugs to underlying target engagement or behavioral efficacy. To clarify these relationships, we examined receptor occupancy measurements using positron emission tomography (PET) (n = 3~5), fMRI (n = 5~8) and a cataleptic behavior (n = 6) with raclopride, a dopamine D2 receptor antagonist (8, 20, and 200 µg/kg) on Wistar rats. Dopamine D2 receptor occupancy was increased dose dependently by raclopride (41.8 ± 2.7%, 8 µg/kg; 64.9 ± 2.8%, 20 µg/kg; 83.1 ± 3.0%, 200 µg/kg). phMRI study revealed significant positive responses to raclopride at 200 µg/kg specifically in the striatum and nucleus accumbens, related to dopaminergic system. Slight fMRI responses were observed at 20 µg/kg in some areas corresponding to the striatum and nucleus accumbens. There were no noticeable fMRI responses at 8 µg/kg raclopride administration. Raclopride at 200 µg/kg significantly increased the cataleptic score, although, at 8 and 20 µg/kg, raclopride had no significant effects. These findings showed that raclopride-induced fMRI responses were observed at doses inducing cataleptic behavior and high D2 receptor occupancy, suggesting that phMRI can be useful for dose selection in clinical trial as an evaluation method of brain activity, which reflects behavioral responses induced by target engagements.

(R)- and (S)-ketamine induce differential fMRI responses in conscious rats.

(R,S)-ketamine exerts robust antidepressant effects in patients with depression when given at sub-anesthetic doses. Each of the enantiomers in this racemic mixture, (R)-ketamine and (S)-ketamine, have been reported to exert antidepressant effects individually. However, the neuropharmacological effects of these enantiomers and the mechanisms underlying their antidepressive actions have not yet been fully elucidated. Therefore, we investigated the effect of (R,S)-, (R)-, and (S)-ketamine on brain activity by functional MRI (fMRI) in conscious rats and compared these with that of N-methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (n = 5~7). We also assessed their pharmacokinetic profiles (n = 4) and their behavioral effects (n = 7~9). This pharmacological MRI study revealed a significant positive response to (S)-ketamine specifically in the cortex, nucleus accumbens and striatum. In contrast, negative fMRI responses were observed in various brain regions after (R)-ketamine administration. (R,S)-ketamine, evoked significant positive fMRI responses specifically in the cortex, nucleus accumbens and striatum, and this fMRI response pattern was comparable with that of (S)-ketamine. MK-801-induced similar fMRI response pattern to (S)-ketamine. The fMRI responses to (S)-ketamine and MK-801 showed differential temporal profiles, which corresponded with brain concentration profiles. (S)-ketamine and MK-801 significantly increased locomotor activity, while (R)-ketamine produced no noticeable change. (R,S)-ketamine tended to increase locomotor activity. Our novel fMRI findings show that (R)-ketamine and (S)-ketamine induce completely different fMRI response patterns on rat, and that the response produced by the latter is similar to that elicited by an NMDAR antagonist. Our findings provide insight into the antidepressant mechanism of (R,S)-ketamine.

Evaluation of hepatic function using dynamic contrast-enhanced magnetic resonance imaging in melanocortin 4 receptor-deficient mice as a model of nonalcoholic steatohepatitis.

INTRODUCTION: Melanocortin 4 receptor-deficient (MC4R-KO) mice fed a high-fat diet (HFD) develop liver pathology similar to human nonalcoholic steatohepatitis (NASH). However, although liver histology and blood biochemistry have been reported, hepatic function has not been evaluated. In the present study, we evaluated hepatic function in MC4R-KO mice fed an HFD using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with gadolinium­ethoxybenzyl­diethylenetriamine pentaacetic acid (Gd-EOB-DTPA). MATERIALS AND METHODS: Wild type (WT) mice and MC4R-KO mice were fed a standard diet (SD) or an HFD for 20 weeks. The hepatic signal intensity was obtained from DCE-MRI images, and relative enhancement (RE), the time to maximum RE (Tmax), and the half-life of RE elimination (T1/2) were calculated. Histopathological analysis was then performed. RESULTS: Histological analysis with nonalcoholic fatty liver disease activity score (NAS) revealed that MC4R-KO mice fed an HFD achieved the NAS of 5. There was moderate fibrosis in MC4R-KO mice fed an HFD. DCE-MRI with Gd-EOB-DTPA showed that Tmax and T1/2 were significantly longer in MC4R-KO mice fed an HFD compared with wild type (WT) mice (Tmax, WT, 3.9 ±â€¯0.4 min; MC4R-KO, 7.4 ±â€¯1.5 min; T1/2, WT, 23.7 ±â€¯1.9 min; MC4R-KO, 62.5 ±â€¯18.5 min). Tmax and T1/2 were significantly correlated with histopathologic score (steatosis vs. Tmax, rho = 0.48, P = 0.04; steatosis vs. T1/2, rho = 0.50, P = 0.03; inflammation vs. Tmax, rho = 0.55, P = 0.02; inflammation vs. T1/2, rho = 0.61, P < 0.01; ballooning vs. T1/2, rho = 0.51, P = 0.03;fibrosis vs Tmax, rho = 0.72, P < 0.01; fibrosis vs T1/2, rho = 0.75, P < 0.01). CONCLUSIONS: MC4R-KO mice fed an HFD developed obesity and NASH. The liver kinetics of Gd-EOB-DTPA were significantly different in MC4R-KO mice fed an HFD from WT mice, and correlated with the histopathologic score. These results suggest that MC4R-KO mice fed an HFD mimic the hepatic pathology and liver function of human NASH, and therefore might be useful for the study of hepatic dysfunction during the fibrotic stage of NASH.

Anterior cingulate cortex connectivity is associated with suppression of behaviour in a rat model of chronic pain.

A cardinal feature of persistent pain that follows injury is a general suppression of behaviour, in which motivation is inhibited in a way that promotes energy conservation and recuperation. Across species, the anterior cingulate cortex is associated with the motivational aspects of phasic pain, but whether it mediates motivational functions in persistent pain is less clear. Using burrowing behaviour as an marker of non-specific motivated behaviour in rodents, we studied the suppression of burrowing following painful confirmatory factor analysis or control injection into the right knee joint of 30 rats (14 with pain) and examined associated neural connectivity with ultra-high-field resting state functional magnetic resonance imaging. We found that connectivity between anterior cingulate cortex and subcortical structures including hypothalamic/preoptic nuclei and the bed nucleus of the stria terminalis correlated with the reduction in burrowing behaviour observed following the pain manipulation. In summary, the findings implicate anterior cingulate cortex connectivity as a correlate of the motivational aspect of persistent pain in rodents.

ACC2 Deletion Enhances IMCL Reduction Along With Acetyl-CoA Metabolism and Improves Insulin Sensitivity in Male Mice.

Intramyocellular lipid (IMCL) accumulation in skeletal muscle greatly contributes to lipid-induced insulin resistance. Because acetyl-coenzyme A (CoA) carboxylase (ACC) 2 negatively modulates mitochondrial fatty acid oxidation (FAO) in skeletal muscle, ACC2 inhibition is expected to reduce IMCL via elevation of FAO and to attenuate insulin resistance. However, the concept of substrate competition suggests that enhanced FAO results in reduced glucose use because of an excessive acetyl-CoA pool in mitochondria. To identify how ACC2-regulated FAO affects IMCL accumulation and glucose metabolism, we generated ACC2 knockout (ACC2-/-) mice and investigated skeletal muscle metabolites associated with fatty acid and glucose metabolism, as well as whole-body glucose metabolism. ACC2-/- mice displayed higher capacity of glucose disposal at the whole-body levels. In skeletal muscle, ACC2-/- mice exhibited enhanced acylcarnitine formation and reduced IMCL levels without alteration in glycolytic intermediate levels. Notably, these changes were accompanied by decreased acetyl-CoA content and enhanced mitochondrial pathways related to acetyl-CoA metabolism, such as the acetylcarnitine production and tricarboxylic acid cycle. Furthermore, ACC2-/- mice exhibited lower levels of IMCL and acetyl-CoA even under HFD conditions and showed protection against HFD-induced insulin resistance. Our findings suggest that ACC2 deletion leads to IMCL reduction without suppressing glucose use via an elevation in acetyl-CoA metabolism even under HFD conditions and offer new mechanistic insight into the therapeutic potential of ACC2 inhibition on insulin resistance.

Evaluation of a filament perforation model for mouse subarachnoid hemorrhage using 7.0 Tesla MRI.

The filament perforation model (FPM) in mice is becoming increasingly popular to elucidate the molecular pathogenesis of neuronal injury after subarachnoid hemorrhage (SAH). We evaluated brain MRI in a mouse FPM. A total of 28 male C57Bl/6J mice were used. Seventeen animals underwent SAH induction by FPM. In two animals, transient middle cerebral artery occlusion (MCAo) was induced. Nine mice served as controls. T1-weighted images (T1WI), T2-weighted images (T2WI), T2(∗)-weighted images (T2*WI) and apparent diffusion coefficient maps were acquired at day 0 and at various time points following SAH (range: day 1-6 after SAH). Cerebral blood flow (CBF) analysis by (14)C-iodoamphetamine ((14)C-IMP) autoradiography was conducted in nine animals. Hemorrhage could be best confirmed using T2*WI. The degree of hemorrhage varied. All animals evaluated for ⩾2days were hydrocephalic, which was best seen on T2WI. T2-hyperintensity of the corpus callosum and external capsule, indicating white matter (WM) injury, was present after SAH. Ventricle and WM injury volumes were statistically significantly higher at day 3 compared to day 0. Territorial ischemia was detectable in MCAo but not in SAH. Markedly hypointense cortical veins were visible in the hyperacute and delayed phase after SAH on T2*WI. The (14)C-IMP analysis indicated decreased CBF after SAH. MRI is feasible and useful in evaluating pathophysiological changes over time. T2*WI seems best for SAH detection and grading. The chronological change of hydrocephalus and WM injury could be analyzed. T2*WI illustrated specific signal changes of cortical veins, possibly caused by increased oxygen extraction fraction due to decreased CBF.

Gd-EOB-DTPA-enhanced-MR imaging in the inflammation stage of nonalcoholic steatohepatitis (NASH) in mice.

OBJECTIVE: The purpose of this study is to investigate the correlation between the liver kinetics of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) and liver histopathology in a mouse model of NASH by using dynamic contrast-enhanced MRI. MATERIALS AND METHODS: Twenty male C57/BL6 mice aged 8weeks were fed a methionine-choline-deficient (MCD) diet for 2, 4 and 6weeks (MCD groups: MCD 2w, 4w, or 6w). Gd-EOB-DTPA-enhanced MR imaging of the liver was performed at 2, 4 and 6weeks after the MCD feeding. The signal intensity of the liver was obtained from dynamic MR images and relative enhancement (RE), and the time to maximum RE (Tmax) and half-life of elimination RE (T1/2) were calculated. After MRI scan, histopathological scores of hepatic steatosis and inflammation and blood biochemistry data, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, were obtained. RESULTS: Plasma AST and ALT levels were significantly increased in mice fed MCD. Histopathological scores indicated that steatohepatitis progressed with the MCD feeding period from 2 to 6weeks, but significant fibrosis was observed only in mice fed MCD for 6weeks. Gd-EOB-DTPA-enhanced MRI showed that Tmax was significantly prolonged in the livers of the 6-week group compared to the control group (control, 4.0±0.7min; MCD 6w, 12.1±1.6min), although there was no alteration in the 2- and 4-week groups. T1/2 was significantly prolonged in mice fed MCD for 4 and 6weeks compared to the control group (control, 19.9±2.0min; MCD 4w, 46.7±8.7min; MCD 6w, 65.4±8.8min). The parameters of Gd-EOB-DTPA kinetics (Tmax and T1/2) in the liver were positively correlated with the liver histopathological score (steatosis vs Tmax, rho=0.69, P=0.0007; inflammation vs Tmax, rho=0.66, P=0.00155; steatosis vs T1/2, rho=0.77, P<0.0001; inflammation vs T1/2, rho=0.73, P=0.0003). CONCLUSIONS: The liver kinetics of Gd-EOB-DTPA correlated well with the inflammation score in the mouse model of NASH, suggesting the possibility of detecting the steatohepatitis stage without fibrosis by Gd-EOB-DTPA-enhanced MR imaging.

Time Course of Diffusion Kurtosis in Cerebral Infarctions of Transient Middle Cerebral Artery Occlusion Rat Model.

OBJECTIVE: To evaluate the relationship between fiber bundle direction and changes in diffusion kurtosis, we evaluated the apparent diffusion kurtosis coefficients (AKCs) that were perpendicular to and parallel to the principal diffusion tensor eigenvector. MATERIALS AND METHODS: Adult male Wistar rats were subjected to 30 or 60 minutes of middle cerebral artery occlusion and imaged with a 7T Magnetic Resonance Imager System (Varian MRI System 7T/210: Agilent Technologies, CA). Diffusion kurtosis images were obtained before middle cerebral artery (MCA) reperfusion and 3, 6, and 24 hours after reperfusion to generate the apparent diffusion coefficient (ADC), fractional anisotropy (FA), mean apparent diffusion kurtosis coefficient (mAKC), AKC axial to the eigenvector (axAKC), and AKC radial to the eigenvector (radAKC) images. The time course of the region/normal ratio was evaluated for the above parameters in the caudoputamen and white matter. RESULTS: Relative FA and relative ADC values decreased 3 hours after MCA reperfusion and remained decreased until 24 hours. Relative mAKC, axAKC, and radAKC values were increased 3 hours after MCA reperfusion, peaked after 6 hours, and slightly decreased after 24 hours. In the white matter, axAKC showed larger changes than radAKC. CONCLUSION: The time course of the diffusion kurtosis value showed earlier pseudonormalization than the ADC value of the lesions. For white matter lesions, the increase in axAKC was larger than that in radAKC, suggesting that the tissue changes after infarction mainly produce reduced diffusivity along the fibers and lead to increased inhomogeneity of the diffusion.

Pharmacological MRI response to a selective dopamine transporter inhibitor, GBR12909, in awake and anesthetized rats.

Pharmacological magnetic resonance imaging (phMRI) is a powerful tool for imaging the effects of drugs on brain activity. In preclinical phMRI studies, general anesthesia used for minimizing head movements is thought to influence the phMRI responses to drugs. In this study we investigated the phMRI responses to a selective dopamine transporter (DAT) inhibitor, GBR12909, and a dopamine (DA) releaser, d-amphetamine (AMPH), in the isoflurane anesthetized and awake rats using a relative cerebral blood volume (rCBV) method. AMPH (1 mg/kg i.p.) caused an increase in rCBV in the dopaminergic circuitry in the both anesthetized and awake rats. The striatal rCBV change was correlated with the change of the striatal DA concentration induced by AMPH in the both anesthetized and awake rats. GBR12909 (10 mg/kg i.p.) caused a positive rCBV response and showed a similar regional pattern of rCBV response to AMPH in the awake rats, and the correlation between the change of the striatal rCBV and the striatal DA concentration was observed. However, in the anesthetized rats, GBR12909 induced a widespread negative rCBV response, whereas an increase in striatal DA concentration was observed. These findings indicate that phMRI responses to activation of DA neurotransmission by GBR12909 or AMPH are overall identical in the awake state, while the phMRI response to a DAT inhibitor, GBR12909 but not to AMPH was changed by isoflurane anesthesia. For the evaluation of neuroactive drugs using phMRI, isoflurane anesthesia might be complicated the interpretation of pharmacodynamic effects of drugs in preclinical studies.

Dynamic contrast-enhanced MRI of the liver in Mrp2-deficient rats using the hepatobiliary contrast agent Gd-EOB-DTPA.

OBJECTIVES: The objective of this study was to compare the hepatic uptake and biliary excretion of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) in control and multidrug resistance-associated protein 2 (Mrp2)-deficient rats by noninvasive dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and the impact of Mrp2 deficiency on organic anion-transporting polypeptide 1 (Oatp1) transporters and liver vascularization by immunohistochemistry. MATERIALS AND METHODS: Twenty rats were used in the normal control (n = 10) and Mrp2-deficient rat groups (n = 10). Dynamic contrast-enhanced magnetic resonance imaging studies were performed using Gd-EOB-DTPA (0.025 mmol Gd/kg; 0.1 mL/kg body weight) as the contrast agent. The percentages of relative enhancement were calculated at each time point after Gd-EOB-DTPA injection. In addition, relative enhancement maps were generated through pixel-by-pixel calculations before the injection and at 5, 10, 20, 30, and 40 minutes after the injection. After the DCE-MRI study, blood was sampled from all rats and 6 blood sample parameters, serum aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids, were measured. Rat livers were processed for histologic diagnosis to clarify contrast agent uptake/efflux by examining Oatp1, Mrp2, and platelet endothelial cell adhesion molecule immunohistochemical staining. RESULTS: The relative enhancement of the Mrp2-deficient, Eisai hyperbilirubinuria rats (EHBRs) (48.6% [3.4%]) was significantly lower than that of the control rats (64.0% [3.2%]; P < 0.001) 5 minutes after the Gd-EOB-DTPA injection. Thereafter, the relative enhancement observed in the EHBRs (10 minutes, 59.6% [5.4%]; 20 minutes, 67.8% [4.1%]; 30 minutes, 69.1% [4.2%]; 40 minutes, 71.0% [4.2%]; P < 0.0001) was significantly higher than that in the control rats at the same time points after the Gd-EOB-DTPA injection. The aspartate aminotransferase and alanine aminotransferase values were not significantly different between the 2 groups. However, total bilirubin, direct bilirubin, indirect bilirubin, and total bile acids levels in EHBRs were significantly higher than those in the control rats. The percentages of the Mrp2-positive cells in the control rats were higher compared with the EHBRs (control, 0.3% [0.1%]; EHBR, 14.1% [3.6%]; P < 0.01). However, the percentages of the Oatp1-positive cells were not different between the 2 groups. Moreover, the percentages of the platelet endothelial cell adhesion molecule-positive cells in the blood vessels of the control rat livers were higher compared with the EHBRs (control, 17.5% [3.3%]; EHBR, 9.5% [3.9%]; P < 0.01). CONCLUSIONS: The utility of noninvasive DCE-MRI with Gd-EOB-DTPA as a tool for the assessment of Mrp2-deficient hyperbilirubinuria rats was demonstrated. We also clarified that the lower vascular density in the EHBRs may cause delayed uptake of the contrast agent compared with the control rats. In addition, the lower Mrp2 transporter expression may cause the lower efflux of the contrast agent from the Mrp2-deficient rats compared with the control rats.

Assessment of peripheral tissue perfusion disorder in streptozotocin-induced diabetic rats using dynamic contrast-enhanced MRI.

PURPOSE: To assess peripheral tissue perfusion disorder in streptozotocin (STZ)-induced diabetic rats by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). MATERIALS AND METHODS: A rat diabetes model was produced by intravenous injection of STZ. Diabetic rats were sustainably treated with either saline or insulin using an Alzet osmotic pump. Hind paw tissue perfusion was measured by signal intensity (SI) enhancement after gadolinium diethylenetriaminepentaacetic acid injection in DCE-MRI study and quantified using the initial area under the SI-time curve (IAUC). Peripheral tissue uptake of [(14)C]iodoantipyrine (IAP) was also determined as a marker of tissue blood flow for comparison with the IAUC value indicating tissue perfusion. RESULTS: STZ caused hyperglycemia at 1 and 2 weeks after injection. Treatment with insulin significantly alleviated hyperglycemia. At 2 weeks after STZ injection, peripheral tissue perfusion was clearly reduced in the diabetic rats and its reduction was significantly improved in the insulin-treated diabetic rats. Tissue perfusion evaluated by DCE-MRI was similar to the tissue blood flow measured by [(14)C]IAP uptake. CONCLUSION: Our findings demonstrated that DCE-MRI can assess peripheral tissue perfusion disorder in diabetes. DCE-MRI could be suitable for noninvasive evaluation of peripheral tissue perfusion in both preclinical and clinical studies. It may also be useful for developing novel drugs to protect against diabetic vascular complications.

Sensitive reduction in 14C-acetate uptake in a short-term ischemic rat brain.

14C-acetate is preferentially taken up by astrocytes, and is a useful tool for measurement of glial metabolism. The aim of this study was to determine the effects of short-term ischemia on 14C-acetate uptake in the rat brain. The middle cerebral artery was occluded for 3, 10, or 30 minutes. Five minutes after reperfusion, rats were injected with 14C-acetate and decapitated 5 minutes later. Radioactivity concentrations in striatum and cerebral cortex were determined by autoradiography. Cerebral blood flow was also measured using 14C-iodoamphetamine. Neuronal cell death was measured by Nissl staining, and expression of monocarboxylate transporter-1 was examined by immunohistochemical staining. A significant reduction of 14C-acetate uptake was observed in striatum by 3 minutes of occlusion. The degree of reduction of 14C-acetate uptake and reduction area were increased with occlusion period. In contrast, within the same region the regional blood flow was increased by 10 minutes of occlusion, suggesting that uptake of 14C-acetate was independent of blood flow. No neural cell death was detected, and no significant alteration of monocarboxylate transporter-1 expression was observed by 30 minutes of occlusion. These results indicate that 14C-acetate uptake is a sensitive marker for glial metabolism in the ischemic rat brain.

Glial metabolic dysfunction caused neural damage by short-term ischemia in brain.

Although several pieces of evidence have indicated that glial cells support neuronal cells in the ischemia-reperfusion brain, the direct contribution of glial cells to cell damage is not well known. The present study was designed to determine whether there are any changes in cell damage after a short-term middle cerebral artery occlusion (MCAO) when glial metabolism is suppressed. Injection of fluorocitrate (FC) or 10 minutes MCAO alone did not produce cell damage. However, 10 minutes MCAO in rats pretreated with FC caused significant cell damage. These data directly demonstrated that inhibition of glial metabolism might increase neuronal vulnerability to even a short-term transient ischemia.

Discrepancy between cell injury and benzodiazepine receptor binding after transient middle cerebral artery occlusion in rats.

We investigated postischemic alterations in benzodiazepine receptor, D1 dopamine receptor, and muscarinic acetylcholine receptor binding after transient middle cerebral artery (MCA) occlusion in rats using [3H]-flumazenil, [3H]-SCH23390, and [3H]-N-methyl-4-piperidyl benzilate ([3H]-NMPB), respectively, as radioligand. These ligand bindings were determined at 3 and 24 h and at 3 and 7 days after ischemia/reperfusion of MCA by using autoradiographic methods. Ischemic cell injury was clearly detected from 3 h after ischemia/reperfusion and progressively increased from 3-24 h after ischemia/reperfusion of MCA. The area of cell injury reached maximum at 24 h after ischemia/reperfusion of MCA. [3H]-SCH23390 binding was reduced to 47% of the contralateral side at 3 days after ischemia/reperfusion of MCA. After 7 days, [3H]-SCH23390 binding was further reduced by 20% in the striatum. [3H]-NMPB binding was slightly decreased in both the striatum and cerebral cortex at 3 days after ischemia/reperfusion of MCA, and [3H]-NMPB binding in the striatum and cerebral cortex were reduced to 42 and 62% of the contralateral side at 7 days after ischemia/reperfusion of MCA. [3H]-NMPB was also decreased at 24 h. In contrast, [3H]-flumazenil binding was not decreased in the striatum and cerebral cortex within 7 days after ischemia/reperfusion of MCA. These results suggest that [3H]-SCH23390 and [3H]-NMPB binding do not correlate with cell injury by ischemia/reperfusion, although vulnerability to ischemia/reperfusion was observed with these receptors. In addition, central benzodiazepine receptor imaging might be essentially stable to neuronal cell injury induced by transient focal cerebral ischemia in rats, in contrast to the results of PET studies.