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Background Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. Methods To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density–weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). Results We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex ( r = 0.6397, P = .0283) and outer stripe of the outer medulla ( r = 0.7810, P = .0039)]. Conclusions By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.

A cream that contains menthol and diphenhydramine is widely prepared in hospital pharmacies and prescribed to patients for the treatment of pruritus associated with chronic kidney disease. However, there is a serious concern regarding its physical stability; therefore, we investigated this issue using magnetic resonance imaging (MRI). For a sample preparation, a menthol-containing ethanol solution was mixed with a commercial diphenhydramine cream. After storage for 7 d at 40°C, substantial phase separation into two distinct layers (upper and lower layers) was observed in the sample. This study further examined the components of the phase-separated layers using magnetic resonance (MR) spectroscopy and chemical shift selective images, and it was verified that the upper layer consisted of packed oil droplet layers, whereas the lower was an aqueous phase. Subsequently, the time-dependent phase separation of the sample at different temperatures was investigated. From the MR images, including a T2 relaxation time map and apparent diffusion coefficient maps, it was obvious that the phase separation developed further with increasing temperature; the most substantial phase separation was observed from the sample stored at 40°C, while no phase separation was detected at 25°C. In the final phase of this study, we conducted a formulation study and succeeded in improving the cream's physical stability by adding a hydrophilic surfactant to the preparation.

A mixed preparation consisting of a water-in-oil emulsion-type moisturizing cream and a steroid ointment is frequently prescribed for the treatment of atopic dermatitis. We have investigated the compatibility of moisturizing creams and ointments because there are concerns regarding the physical stability of these mixed preparations. The key technology used in this study was magnetic resonance imaging (MRI). A commercial moisturizing cream and white petrolatum or clobetasone butyrate (CLB) ointment samples were mixed in a weight ratio of 1 : 1. A centrifugation test protocol (20000×g for 3 min) was implemented to accelerate the destabilization processes in the samples. After centrifugation, the mixed preparations separated into three distinct layers (upper, middle, and lower), while no phase separation was observed using moisturizing cream alone. The phase separation was monitored using chemical shift selective images of water and oil and quantitative T2 maps. In addition, MR and near-infrared spectroscopy were employed for component analysis of each phase-separated layer. Collectively, it was confirmed that the lower layer contained water, oils, and organic solvent, while the upper and middle layers were composed solely of oils. Furthermore, this study investigated the distribution of CLB in the phase-separated samples and showed that a heterogeneous distribution existed. From our results, it was confirmed that the mixed preparation became unstable because of the incompatibility of the moisturizing cream and ointment.

Renal fibrosis (RF) is an indicator for progression of chronic kidney disease (CKD). Although diabetic nephropathy (DN) is the leading cause of CKD and end-stage renal disease in Western populations, the ability of MRI to evaluate RF in DN patients has not been determined. As a first step to identify possible MRI methods for RF evaluation, we examined the use of diffusion tensor imaging (DTI) MRI to evaluate RF in a rat model of DN (SHR/NDmcr-cp(cp/cp): SHR/ND). The signal-to-noise ratio in DTI MRI was enhanced using a spin-echo sequence, and a special kidney attachment was developed for long-term stabilization. The changes in renal temperature and blood flow during measurement were minimal, suggesting the feasibility of this method. At 38 weeks of age, RF had aggressively accumulated in the outer stripe (OS) of the outer medulla. FA maps showed that this method was successful in visualizing and evaluating fibrosis in the OS of the SHR/ND rat kidney (r = 0.7697, P = 0.0126). Interestingly, in the FA color maps, the directions of water molecule diffusion in RF were random, but distinct from conventional water diffusion in brain neuron fibers. These findings indicate that DTI MRI may be able to evaluate RF in CKD by DN.

The effects of non-noxious skin stimulation on nerve growth factor (NGF) secretion in the parietal cortex were examined in anesthetized rats. Innocuous skin stimulation was delivered to the left hindlimb with a soft-hair brush. Extracellular NGF in the right parietal cortex was collected by microdialysis methods using a protein-permeable probe and was measured using an enzyme-linked immune-sorbent assay. Brushing produced a significant increase in extracellular NGF levels. This NGF response was not observed in rats pretreated with a nicotinic ACh receptor (nAChR) antagonist mecamylamine. We further examined whether brushing could activate the basal forebrain nucleus (nucleus basalis of Meynert, NBM), which is the main source of cholinergic fibers in the cerebral cortex, by means of functional MRI. The blood oxygen level-dependent signal in the right NBM was significantly higher during brushing compared to baseline. The results suggest that non-noxious skin stimulation activates NBM and promotes NGF secretion in the parietal cortex via nAChRs.

MRIを診断に応用する技術はここ20年間で大きく進歩した．無侵襲で体の軟部組織の病態変化を空間分解能高く画像化できることがMRIの特徴であり，内臓，脊髄や脳などの診断に適用されて今ではなくてはならないものになった．この方法を創薬研究に応用したものがphMRIである．製薬業界では中枢疾患治療薬の需要が高まっているにも関わらず，創薬にかかる莫大な費用と病態や治療における客観的なバイオマーカーが不足していることが原因で，新薬を容易に生み出せない状況が続いていた．このような状況の打開策としてMRIの1つの重要な方法であるfMRIを使った創薬バイオマーカー探索技術が進歩しはじめた．この方法によりヒトを含めた動物の脳機能をいろいろな側面で客観的に評価できることから，創薬バイオマーカーに対しfMRIは非常に有効であることが実証されphMRIと呼ばれている．具体的には薬品開発プロセスの異なったステージにまたがるようなターゲットバリデーション，中枢におけるオフターゲット効果，副作用ならびに治療濃度域の評価など早期の中枢への効果，早期のコンセプトの評価，患者の層別化，ドージングの最適化（投薬計画），レスポンダー識別等に関しphMRIによる信頼性の高いバイオマーカーが得られている．FDAがCPI白書で動物試験に画像法を用いることを推奨しており，創薬研究パイプラインの様々なポイントへ実験用MRIの導入が増えている．特に薬効薬理試験をはじめ毒性試験への応用が盛んになってきている．phMRIの使用が盛んになればより多くの信頼できるバイオマーカーが発見され，多くの有効な中枢治療薬の開発が促進されるだろう．また，様々なフェーズの精神疾患患者の脳機能と投薬の関係を詳細に調べることが可能になることを意味しており，まさに精神疾患の予防法の開発やテーラーメード医療が現実になる日が遠くないことを示している．

To investigate whether dynamic and delayed magnetic resonance imaging (MRI) with gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA), a hepatobiliary MRI contrast agent, has potential for the differential diagnosis of experimental hepatic tumors.Twelve male rats received N-nitrosomorpholine solution as drinking water to induce hepatic tumors. After injection of Gd-EOB-DTPA, rats were subjected to dynamic and delayed MRI. The relative enhancement (RE) was calculated, and the time of the maximum RE (Tmax) was evaluated. After MRI, liver was histologically analyzed.One hundred sixty-three hepatic tumors 3-12 mm in diameter were induced after 18 weeks of treatment with 0.01 wt/vol% of N-nitrosomorpholine, and 81 of them were evaluated. The RE in hyperplastic nodules (HPNs) was significantly higher than that in moderately or poorly differentiated hepatocellular carcinomas (HCCs) in the late phase, whereas there was no significant difference in RE between well-differentiated HCCs and HPNs. The average Tmax in HPNs was about 13 minutes, whereas that of each differentiated HCCs was about 1 minute.It was possible to differentiate benign HPNs and malignant HCCs (especially well-differentiated HCCs) by evaluating the change of RE or comparison of Tmax with Gd-EOB-DTPA-enhanced MRI.

Kato N, Yokawa T, Tamura A, et al. Gadolinium-ethoxybenzyl-diethylenetriamine-pentaacetic acid interaction with clinical drugs in rats. Invest Radiol 2002;37:680–684. To investigate whether the hepatic enhancement characteristics of Gd-EOB-DTPA are influenced by the preapplication of a variety of commonly used clinical pharmaceuticals (eg, antibiotics, antineoplastic drugs, corticosteroids, antiarrhythmia drugs, antianxiety drugs, scopolamine, and xanthine derivatives). Eleven commercially available drugs (prednisolone, rifampicin, doxorubicin hydrochloride, cisplatin, propranolol hydrochloride, scopolamine butylbromide, theophylline, ampicillin, cefotaxime sodium, verapamil hydrochloride, and diazepam) were intravenously (IV) injected in rats at three to five times the clinical dose (n = 3 or 6 per drug). A control group of rats was given saline (n = 6). Gd-EOB-DTPA (25 μmol Gd/kg IV) was administered to rats 30 minutes after the injections of the clinical drugs. Liver MR imaging was performed with a 2.0 T animal imager before and up to 60 minutes after injection. Enhancement (ENH) (%) and area under the data from time versus enhancement curve (AUD) were calculated. Rifampicin was the only drug that significantly decreased the hepatic enhancement by Gd-EOB-DTPA. Both the maximum enhancement of the liver and the AUD were significantly reduced when rifampicin was preinjected. Preinjection of prednisolone, doxorubicin hydrochloride, cisplatin or propranolol hydrochloride yielded a slightly but significant increased maximum enhancement of the liver. Furthermore, the enhancement declined more slowly when these drugs were preadministered, yielding a large AUD. None of the other drugs showed a significant effect on hepatic enhancement. Rifampicin exerted a clinically significant decrease on hepatic enhancement by Gd-EOB-DTPA. Prednisolone, doxorubicin hydrochloride, cisplatin, or propranolol hydrochloride slightly but significantly increased the hepatic enhancement by Gd-EOB-DTPA.

A series of new diethylenetriaminepentaacetic acid (DTPA)-bisamide chelators has been prepared and characterized for application as zinc sensors. We have designed and synthesized (GdL(a))(2-), which contains a DTPA-bisamide moiety. The R(1) relaxivity of (GdL(a))(2-) solution decreased monotonically on the addition of Zn(2+). Moreover, (GdL(a))(2-) showed high selectivity for Zn(2+) against Ca(2+) and Mg(2+). We also measured the UV-visible spectra and the coldspray ionization (CSI) MS spectra and concluded that the 1-to-1 Zn(2+) complex of (GdL(a))(2-) is stable at higher concentrations of Zn(2+). These complexes should provide the basis for creating a superior Zn(2+)-sensitive MRI contrast agent and are excellent candidates for incorporation into sensors designed for selective detection of Zn(2+) in biological applications.

Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4-mRNA is increased in the peri-infarcted cortex during the observation period ( approximately 7 days) after MCA-occlusion, maximally on day 3. The change corresponds to the generation and resolution of brain edema monitored by MRI. The signals for the mRNA are predominantly observed in glial cells in the molecular and outer granular layer of the peri-infarcted cortex. These results indicate that AQP4 plays a role in post-ischemic edema formation.

Each L-amino acid (AA) in plasma and brain remains unchanged while normal diet is available. Once L-lysine (Lys) deficient diet was offered to rats, Lys in plasma and brain declined, and anorexia occurred. When solutions of AAs were offered, they selected the Lys solution, and their food intake and growth normalized. The single neuron activity in the lateral hypothalamic area of these rats suggested that neural plasticity occurred, specifically responding to Lys, both by iontophoretic application and during ingestion of AA. The recognition site for deficient nutrient intake in the brain of rats with Lys deficiency was identified by non-invasive magnetic resonance imaging (MRI 4.7 tesla, 40 cm bore in diameter) developed to monitor changes in cerebral blood flow and oxygenation in rat brain. Wistar strain young male rats fed with Lys deficient diet for 4 days, were adapted to be settled in the center of the bore. When they received a Lys injection intraperitoneally (0.2 M, 10 mL/kg), a signal intensity decrease in the medial and lateral hypothalamus appeared 30 minutes later in T2* weighted images, reflecting increased oxygenation which lasted for 30 minutes, and then gradually recovered. These changes never occurred in any other areas of the brain of rats with Lys deficiency, i.e., the thalamus, the cortex, the hippocampus, etc. There were no changes in the signal intensity with control injection of saline. In addition, oxygen comsumption in the brain of rats without Lys deficiency was not altered by intraperitoneal Lys injection. The present results suggest that in essential AA deficiency, the medial and lateral hypothalamus may play important roles in recognition responses to particular deficient nutrients in order to maintain homeostasis.

The main objective of this study was to assess cardiac death (CD) kidney grafts before transplantation to determine whether blood oxygen level-dependent (BOLD) and diffusion MRI techniques can predict damage to these grafts after transplantation. We assessed CD kidney tissue by BOLD and diffusion MRI. We also examined pathological and gene expression changes in CD kidney grafts before and after transplantation. Although there was significantly more red cell congestion (RCC) in the inner stripe of the outer medulla (IS) in both 1 h after cardiac death (CD1h) and CD2h kidneys destined for grafts before transplantation compared with CD0h (p

An intelligent MRI contrast agent has been designed by taking advantage of a microenvironmental responsive polyion complex (PIC). The intelligent signal switching was achieved by controlling Gd ion-water interactions in the microenvironmental responsive PIC. MRI signal of the agent appeared only in tumor region, selectively, and did not appear in normal tissues, when the agent was administrated to a tumor-bearing mouse from the tail vein. The acidic microenvironment, such as lower extracellular fluid pH, higher expression of acidic proteins and polysaccharides, was suggested to cause the tumor-selective activation of the agent.

Transcranial direct current stimulation (tDCS) is one of the noteworthy noninvasive brain stimulation techniques, but the mechanism of its action remains unclear. With the aim of clarifying the mechanism, we developed a rat model and measured its effectiveness using fMRI. Carbon fiber electrodes were placed on the top of the head over the frontal cortex as the anode and on the neck as the cathode. The stimulus was 400- or 40-μA current applied for 10 min after a baseline recording under an anesthetized condition. The 400-μA stimulation significantly increased signal intensities in the frontal cortex and nucleus accumbens. This suggests anodal tDCS over the frontal cortex induces neuronal activation in the frontal cortex and in its connected brain region.

Our previous study showed that injection of the hydrogel of a water-absorbent polymer (WAPGEL) into the ventromedial nucleus of the hypothalamus (VMH) induced stereotyped long-lasting running activity without association of any aggressive reactions, and suggested that swelling of the polymer at the site of injection affected the inhibitory neuron system, resulting in the expression of running activity. In the present study, we injected WAPGEL impregnated with different doses of GABA. By this treatment, the expression of running activity was inhibited in a dose-dependent manner. Contrarily, microinjection of a GABA-A receptor blocker, bicuculline methiodide (BM), released the running reaction in a dose-dependent manner with similar features to that induced by WAPGEL. It is suggested that the running-related neurons in the VMH are inhibited by GABA when the animals are at rest.

Spatio-temporal dynamics of activated brain areas induced by drinking were investigated and visualized in behaving rats using functional magnetic resonance imaging (fMRI). The rats were trained to drink in the magnet bore, and the images were taken during and after drinking glucose and distilled water. During glucose ingestion, the signal intensity was increased continuously and maximally in the lateral hypothalamic area (LHA) and the ventromedial hypothalamus (VMH). Somewhat less intense activation in the central nucleus of the amygdala (AMc), and transient activation in the piriform cortex and the mediodorsal nucleus of the thalamus were observed. The signal intensities of other regions measured were largely unchanged. During post-ingestive periods, the signals re-increased in the hypothalamic areas and AMc. When water was given, LHA and VMH were similarly activated, however, the signal intensity in the amygdala was not significantly increased. The results indicate that these brain regions are activated differentially during drinking behavior, and that LHA and VMH play a central role in the control of not only feeding but also drinking. The regional activities in LHA and VMH are not principally related to the gustatory sensation, and the reactivation after drinking may be related to satisfaction or post-ingestive nutritional information. Also, the responses of AMc are probably due to reward value difference. To the best of our knowledge, this is the first report of mapping of brain areas using fMRI in behaving rats. The improved method described in this study for collecting fMRI data in behaving animals will be useful for studying functional network during animal behavior.

The conventional MRI contrast agents consisting of a chelate complex with a Gadolinium (Gd) ion, such as Magnevist® (Schering AG, Germany), enhance MRI signal by shorting the longitudinal relaxation time of protons of surrounding water molecules (1). These agents have been used as a routine clinical tool to reveal anatomical details in vivo and to detect lesions for diagnosis. For the future imaging of molecular/cellular diagnosis (2), innovative agents having much stronger proton relaxivity should be required. Since their discovery in 1985 (3), potential applications for fullerene and metallofullerene materials have been proposed because of their unique structural and electronic properties. Biomedical application, especially as MRI contrast, X-ray or radiopharmaceutical agents, should be a prominent field because fullerene cage can encage various metal ions, such as Ca, Sc, Sr, Y, La, Ce, Sm, Gd, Dy, Er, Yb, Ho, etc. (4). Here, we have investigated the possibility of application of water-soluble Gd metallofullerene for MRI contrast agents, and have evaluated them in vivo as well as in vitro.

To alter neural networks in a restricted area of the brain, we previously developed the hydro-polymer gel (PG) implantation technique. In this study, we found that bilateral or unilateral injection of PG (1.6 μl) into the ventromedial nucleus of the hypothalamus (VMH), contrary to injections into the preoptic area (POA) or posterior hypothalamic area (PHA), severely attenuated circadian changes of locomotor activity in blinded female rats. In addition, PG injection into the VMH also suppressed circadian changes in serum melatonin levels and induced persistent estrus. The effect of PG injection into the VMH on free-running rhythm was blocked by a complete cut around the VMH or a dorsal cut of the VMH but not by anterior or posterior cuts of the VMH. These results suggest that PG injection into the VMH induces some form of neural output via a dorsal route of the VMH that affects the generation of circadian activity rhythm.

Appetite for food, and taste preferences for amino acids and NaC1 in animals are affected by the state of protein nutrition [1–4]. Past experience is also thought to be important for food selection. Rats in a nutritionally well-balanced condition prefer monosodium l-glutamate (MSG) and l-arginine solutions to NaC1 and other amino acids, this preference being directed toward l-lysine and NaCl when the animals are fed lysine-deficient diets. The central site at which preference is regulated, however, is unknown. One of the most important brain sites may be the lateral hypothalamic area (LHA). Some LHA neurons respond during feeding and to anticipatory cues after appropriate conditioning. The ingestion response of the monkey LHA is modulated both by palatability of food and satiation. Some rat LHA neurons respond selectively to the ingestion of lysine during lysine deficiency, but this does not occur in lysine-sufficient controls [4]. To evaluate the role of the LHA in the regulation of the rat preferences in different nutritive conditions, we recorded single neuron activity from the LHA during cue tone presentation and ingestion of various amino acids or NaC1, and compared the responses in controls with those in lysine-deficient rats that had learned to select amino acids and NaCl.

We have previously shown that blockade of neurotransmission mediated by γ-aminobutyric acid (GABA) in the ventromedial nucleus of the hypothalamus (VMH) by the microinjection of a GABAA receptor antagonist, bicuculline methiodide (BM), exclusively induced running activity in the rat. The purpose of the present study was to examine the role of receptors for excitatory amino acids (EAAs) in the VMH in inducing hyper-running and to clarify the interaction between GABA and EAAs in the VMH in controlling running activity. Although the injection of neitherN-methyl- d -aspartate (NMDA) nor quisqualate into the VMH induced hyper-running, kainate (KA) produced running activity in a dose-dependent manner with similar features to that induced by BM. This effect of KA was blocked by a non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX). GABA injected simultaneously with KA into the VMH failed to affect hyper-running induced by KA. On the other hand, DNQX significantly suppressed the BM-induced running activity. These results suggest that endogenous EAAs acting on the KA-type receptor in the VMH facilitates running activity and that the release of such EAAs from the nerve terminal is presynaptically inhibited by GABA.

We have developed a new technique for altering neural construction by implanting self-swelling, the hydrogel of water-absorbent polymer (WAPGEL) into restricted areas of the brain. Using this technique, we found that the VMH is responsible for expression of running activity. Implantation of 1.5 microliters of WAPGEL into the dorsomedial portion of the ventromedial nucleus of the hypothalamus (VMH) of the rat specifically induced bursts of running, whereas implantation into other sites of hypothalamus, e.g., the anterior, lateral and posterior areas, and the dorsomedial nucleus, caused no significant running activity. The WAPGEL-injected rat showed less aversive reactions than the sham-operated rat. Since deafferentation of the VMH after WAPGEL injection prevented the induction of running activity, the output from the VMH appeared to be responsible for the running activity.

According to the Aschoff's role, exposure to continuous light (LL) results in the elongation of the free-running period of the rat circadian rhythm. However, the LL may not always mean the constant intensity of the light for the suprachiasmatic nucleus, since the rat may regulate the contrast of the illumination by their eyelids which are closed during the sleep phase. In this study, the surgical removal of the eyelids under the LL caused arrhythmicity of the locomotor activity in 7 of 10 rats. The remaining 3 rats maintained the free-running rhythm after the removal of the eyelids. These results suggest that constant light may affect the free-running rhythm of the rat with or without eyelids in the different manner.

Exposure to continuous light (LL) results in elongation of the free-running period of the rat circadian rhythm. However, the LL may not always mean constant intensity of light for the suprachiasmatic nucleus (SCN), since rats may regulate contrast of illumination by their eyelids which are closed during sleep phase. The removal of eyelids caused shortening of the free-running period or arrhythmicity under LL. In addition, implantation of carbachol--acetylcholine agonist--near the SCN also resulted in similar effects.