Azabu University Science Information Repository / 麻布大学学術情報リポジトリ
Not a member yet
1842 research outputs found
Sort by
Excretory urography can track down morphological changes in the urinary bladder associated with urachal anomalies in calves for early diagnosis
Get PDFOBJECTIVE
To investigate radiographic detection by excretory urography of morphological changes in the urinary bladder associated with urachal anomalies in calves.
METHODS
Excretory urography was performed to detect morphological changes in the urinary bladder of 13 calves, of which 6 were nondysuric with swelling of umbilical region and 7 were dysuric without clinical umbilical swelling from November 2022 through April 2024.
RESULTS
The urinary bladder was delineated in all 13 calves after excretory urography. The aspect ratios (length:height), which objectively evaluate the shape of the urinary bladder, ranged from 1.08 to 2.43 (1.90 in average) and 1.34 to 11.89 (4.75 in average) in nondysuric and dysuric calves, respectively. The ratios of calves with nondysuric were significantly lower than those of dysuric (P < .05).
CONCLUSIONS
Excretory urography could play an important role in evaluating abnormal morphological changes due to urachus anomalies in the urinary bladder of calves.
CLINICAL RELEVANCE
Among calves with dysuria, urachal anomaly should be included in the differential diagnosis. Excretory urography is proposed as an alternative option for early diagnosis among calves presenting with dysuria to improve livestock productivity.journal articl
Naringenin Suppresses the Hyperexcitability of Trigeminal Nociceptive Neurons Associated with Inflammatory Hyperalgesia: Replacement of NSAIDs with Phytochemicals
Get PDFThe present study examines whether the systemic application of naringenin (NRG) reduces inflammation-induced hyperexcitability in the spinal trigeminal nucleus caudalis (SpVc) related to hyperalgesia, and compares its impact with that of diclofenac (DIC). To provoke inflammation, the whisker pads of rats were injected with complete Freund’s adjuvant, and subsequently, mechanical stimuli were administered to the orofacial region to determine the escape threshold. Compared to naïve rats, the inflamed rats showed a significantly lower mechanical threshold, and this reduced threshold returned to normal levels two days post-administration of NRG, DIC, and half-dose DIC plus half-dose NRG (1/2 DIC + 1/2 NRG). Using extracellular single-unit recordings, the activity of SpVc wide-dynamic range neurons was measured in response to mechanical stimulation of the orofacial area under anesthesia. The average firing rate of SpVc neurons when exposed to both non-painful and painful mechanical stimuli was significantly reduced in inflamed rats following NRG, DIC, and 1/2 DIC + 1/2 NRG administration. The heightened average spontaneous activity of SpVc neurons in rats with inflammation was significantly reduced following NRG, DIC, and 1/2 DIC + 1/2 NRG administration. The increased average receptive field size observed in inflamed rats reverted to normal levels after either NRG, DIC, or 1/2 DIC + 1/2 NRG treatment. These findings indicate that NRG administration can reduce inflammatory hyperalgesia linked to the heightened excitability of SpVc wide-dynamic range neurons.journal articl
Pathological study of proximal tubule mitochondria in diclofenac-induced acute kidney injury model mice
Get PDF麻布大学博士(学術)Diclofenac (DF) is one of the phenylacetic acid non-steroidal anti-inflammatory drugs (NSAIDs) and is frequently prescribed as an antipyretic analgesic and anti-inflammatory drug. It has been reported that adverse drug reaction include targeting mitochondria and inducing nephrotoxicity via reactive oxygen species. However, there are few reports that have analyzed in detail the relationship between mitochondrial changes and nephrotoxicity using pathological methods. Furthermore, when kidney tissue is used as a material for experiments, for example, when analyzing protein expression by Western blotting, it is extremely difficult to classify and use renal tubules in the kidney cortex. Furthermore, in research on changes in mitochondrial membrane potential using cultured cells, renal tubules have not been classified and subjected to experiments. In acute kidney injury (AKI), the proximal tubule has been reported to be particularly vulnerable to mitochondrial dysfunction due to mitochondrial oxidative status because of its dependence on aerobic metabolism. The purpose of this study was to focus on the relationship between autophagy and the proximal tubule, and to compare and examine the process of histological changes time dependence. In addition, we will conduct a detailed immunohistochemical and ultramorphological analysis of mitochondria in the proximal tubules damaged by DF toxicity. This study suggested that DF-induced AKI may shift to damage to the entire renal cortex via autophagy after proximal tubular damage. This study provides extremely important information to support the prevention of DF-induced AKI by maintaining proximal tubule homeostasis in the early stages.
The outline of this research is as follows.
Establishment and biochemical analysis of AKI mouse model
For establishment a DF-induced AKI mouse model, 6-week-old female ICR mice were divided into a control group (n = 3) and 6, 12, and 24-hour post-DF treatment groups (n = 3 each), and the control group received saline. Mice in the DF treatment group were intraperitoneally inoculated once with DF at a dose of 250 mg/kg, and blood and kidneys were collected from the mice in each group after treatment (Azabu University Animal Care and Use Committee: approval number 170524). -2). In serum biochemical analysis, biochemical serum urea nitrogen (BUN) was measured as an indicator of renal dysfunction. As a result, those BUN increased in a time-dependent manner, with the highest value at 116.3 mg/dL in the 24-hour group.
Histological examination
Hematoxylin and eosin (HE) stained specimens of kidney tissue from each group were observed using a light microscope. Ten fields of view were randomly selected at 400x magnification, the number of vacuolated tubules was counted, and the degeneration was classified into 3 stages: mild, moderate and severe based on the degree of degeneration. This operation was repeated 5 times, and the number of vacuolated renal tubules was semi-quantitatively evaluated. Concerning renal tubular damage characterized by vacuolar degeneration of renal tubules in the DF treatment group, an increase in the number of degenerated cells was observed in the 6, 12, and 24-hour groups, indicating significant time-dependent renal damage.
Immunohistochemistry
Immunohistochemical detection of p62, cytochrome c oxidase polypeptide IV (COX IV), 8-nitroguanosine, microtubule-associated protein 1 (MAP1) light chain 3 (LC3), and lysosomal membrane-associated protein 1 (LAMP-1) was performed in renal tissues. For antigens other than p62, serial sections were used to confirm the localization of positive reactions for two antigens (COX IV and 8-nitroguanosine, LC3 and LAMP-1) in the cytoplasm of the same renal tubular cells. A semi-quantitative evaluation was performed using Tukey's multiple comparison test and statistical analysis was performed.
These results showed that mitochondrial damage in the proximal tubules was associated with an increase in the number of COX IV-positive granules in the cytoplasm of the tubules of DF-treated mice, and an increase in the number of COX IV-positive granules in the 12- and 24-hour groups compared to the 6-hour group. An increase in many positive granules was observed in the proximal tubule. Furthermore, the immunostaining scores showed a significant increase in the 12- and 24-hour groups compared to the control group, and COX IV and 8-nitroguanosine-positive granules were approved at approximately the same location within the cytoplasm in degenerating proximal tubular cells. Immunohistochemical analysis of p62 in proximal tubular epithelial cells was performed. There were many p62-positive proximal tubules in the 12-hour and 24-hour groups. More positive cells were observed in the 12-hour group than in the 24-hour group. An increase in the number of both LC3– and LAMP-1–positive granules was also observed in the cytoplasm of renal epithelial cells; large numbers of granules were present in degenerated renal tubules in the 12- and 24-h groups. In LC3, as with p62, more positive cells were observed in the 12-hour group than in the 24-hour group. Analysis of serial sections of renal tissues showed co-localization of LC3– and LAMP-1–positive granules.
Western blotting
Western blot analysis was performed for LC3-I (16 kDa) and LC3-II (14 kDa) using frozen renal cortex. In addition, analysis of blotting images using ImageJ software revealed a significant increase in LC3-I expression in the samples from the 12- and 24-h treatment groups compared with the level of expression in the control samples.
Electron microscopic findings of proximal tubules
Electron microscopic findings of the renal cortex showed more mitochondrial degeneration, swelling, and fragmentation in the DF-treated group than in the control group. Mild mitochondrial degeneration was observed in the 6-hour group, but severe mitochondrial degeneration was markedly observed in the 12- and 24-hour groups. It was also confirmed that most of the degenerated mitochondria in the 12 and 24-hour groups were fragmented. Autophagosomes containing degenerated mitochondria were not observed in the 6-hour group, but were significantly observed in the 12-hour group. Furthermore, in the 24-hour group, the number of autophagosomes decreased, and many fragmented and degenerated mitochondria were observed.
NSAIDs induce AKI by altering renal hemodynamics through inhibition of cyclooxygenase in the arachidonic acid cascade and causing renal structural changes indicative of renal damage, such as brush border loss, tubular dilatation, and progressive lesion formation. DF has been reported to target mitochondria, induce nephrotoxicity through ROS production, and cause oxidative stress and significant DNA fragmentation. In recent reports, mitochondrial membrane potential, mitophagy and mitochondrial fragmentation have been studied using cultured cells and the whole cortex of kidney tissue as material. However, in this study, we focused on proximal tubule, which is one of the targets of damage caused by DF. The pathological examination focused on the renal tubules. DF-induced oxidative stress causes DNA damage, and fragments of damaged DNA can be localized as nitrated nucleobases in organelles, nuclear or mitochondrial DNA, and COX IV-positive cytoplasmic granules was interpreted as indicating damaged mitochondrial DNA in immunohistochemistry. It revealed that COX IV and 8-nitroguanosine have nearly identical localization within the same cell in degenerating proximal tubular cells of DF-treated mice in Immunohistochemical analysis. Furthermore, immunohistochemical analysis of autophagy-related antigens (p62, LC3, LAMP-1) revealed that autophagy activity reached its maximum 12 hours after DF treatment. In addition, electron microscopy findings showed that 12 hours after DF treatment, autophagy (mitophagy) that processes damaged mitochondria was clearly observed, and at 24 hours, the mitophagy pattern decreased, and instead damaged and fragmented mitochondria were observed. This result was consistent with the results of immunohistochemical analysis. On the other hand, Western blotting results for LC3-I and II showed that autophagy activity was highest at 24 hours. As mentioned at the beginning, this is because when collecting Western blotting samples, tissues other than the proximal tubule, such as the distal tubule and glomerulus, are included. In this study, we focused our analysis on the proximal tubule of the renal cortex, and found that injury occurs in the proximal tubule at an earlier stage than 24 hours. It was confirmed that DF toxicity causes mitochondrial damage and fragmentation in the proximal tubule, along with activation of mitophagy, an autophagy mechanism that removes damaged mitochondria. Furthermore, autophagy activity decreased at 24 h compared to 12 h, suggested that it stops processing damaged mitochondria and causes further damage to proximal tubule cells due to accumulation of damaged mitochondria after reduced the clearance capacity of mitophagy.
Recently, research on mitochondrial dysfunction has emerged as an exciting new field for identifying treatments for AKI. Inhibition of mitochondrial fragmentation leads to inhibition of apoptosis, inhibition of mitochondrial fission factor maintains mitochondrial homeostasis in ischemic kidney injury model mice, and renal ischemic preconditioning. It has been reported that Fundc1 is involved in regulating mitochondrial fission when mitophagy is activated during IPC, and mitochondrial fragmentation plays an important role in AKI. Ultramorphological findings also showed fragmented mitochondria in the cytoplasm of the proximal tubules of the DF treatment group at 12 and 24 hours. This study is very important as a report for targeting and analyzing proximal tubular mitochondria to protect or ameliorate DF-induced renal damage.
Many studies have reported the occurrence of renal damage 24 hours after treatment with DF. In this study, the results of serological analysis and Western blotting analysis showed a time-dependent exacerbation of DF-induced renal damage up to 24 hours after DF treatment. This was thought to reflect damage to the entire renal cortex, including tubules other than the proximal tubule. On the other hand, because of focusing pathologically on the proximal tubule, we confirmed that autophagic activity reaches its peak 12 hours after DF treatment, with clear evidence from statistical analysis of histological data and, electron microscopic findings. At the same time, after 24 h of DF treatment, autophagy activity decreased, and electron microscopy showed many damaged mitochondria and fragmentation. As p62, LC3, and LAMP-1 showed maximum values at 12 hours, it became clear that autophagy activity was high at 12 hours, earlier than at 24 hours. This study shows that damage to the entire renal cortex may be exacerbated by stagnation of mitophagy or accumulation of damaged mitochondria in the proximal tubule. As a future application for AKI prevention, it was suggested that DF-induced AKI may be alleviated by maintaining mitochondrial homeostasis in the proximal tubule at an early stage.ジクロフェナク（DF）はフェニル酢酸系非ステロイド性抗炎症薬（Non-Steroidal Anti-Inflammatory Drugs: NSAIDs）の一つで、解熱鎮痛薬および抗炎症薬として最も頻繁に処方されている。副作用にミトコンドリアを標的とし、活性酸素種を介して腎毒性を誘発することが報告されている。しかし、ミトコンドリアの変化と腎毒性の関連について病理学的手法により詳細に分析した報告は少ない。しかも、腎組織を材料として実験に用いる場合、例えばウェスタンブロッティングによるタンパク発現の解析法では、腎の皮質における尿細管を分類して用いることは非常に厳しい。また、培養細胞によるミトコンドリアの膜電位の変化に関する研究においても尿細管を分類して実験に供することは行われていない。急性腎傷害（acute kidney injury: AKI）において、近位尿細管は好気性代謝に依存しているため、ミトコンドリアの酸化状態によるミトコンドリアの機能不全に関して特に脆弱であると報告されている。本研究の目的は、尿細管の中でも特に近位尿細管とオートファジーの関係性に着目し経時的に組織学的変化の過程を比較検討することである。また、DFの毒性により損傷した近位尿細管のミトコンドリアについて免疫組織化学的ならびに超微形態学的により詳細に解析することである。本研究によりDF誘発によるAKIは近位尿細管損傷後にオートファジーを介して腎皮質全体の損傷に移行する可能性が示唆された。初期段階における近位尿細管の恒常性を維持することでDF誘発性AKIの予防につなげるための裏付けとして、本研究は非常に重要な情報となる。
本研究の概要は以下のとおりである。
AKIマウスモデルの確立および生化学的解析
DF誘発AKIマウスモデル確立のため、6週齢雌ICRマウスを対照群 （n= 3）、DF処置後6、12および24時間群（各 n=3）に分け、対照群には生理食塩水を、DF処置群のマウスにはDFを250 mg/kgの用量で1回腹腔内接種し、処置後各群のマウスから血液および腎臓を採材した（麻布大学動物実験委員会：承認番号170524-2）。血清生化学的分析では、腎機能障害の指標として生化学的血清尿素窒素（BUN）を測定した。結果として時間依存的にBUNが増加し、24時間群において116.3mg/dLと最も高値を示した。
組織学的検査
各群の腎組織におけるヘマトキシリン・エオジン（HE）染色標本の観察を光学顕微鏡にて行った。倍率400倍で無作為に10視野選択し、空胞変性尿細管数を数え、変性の程度に基づいて、軽度、中等度および重度の3段階に分類した。この操作を5回行い、空胞変性尿細管数の半定量的評価を行った。DF処置群における尿細管の空胞変性を特徴とする尿細管損傷について6、12および24時間群の順に変性細胞数の増加が認められ、有意な時間依存性の腎損傷が示された。
免疫組織化学
腎組織においてp62、シトクロム c オキシダーゼ ポリペプチド IV （COX IV） 、8-nitroguanosine、微小管関連タンパク質1（MAP1） 軽鎖 3（LC3） およびリソソーム膜関連プロテイン1（LAMP-1）の免疫組織化学的検出を行った。 また、p62 以外の抗原については連続切片を使用し、同じ尿細管細胞の細胞質における2抗原の陽性反応（COX IV および 8-nitroguanosine、LC3および LAMP-1） の局在を確認した。半定量的評価を行い、Tukeyの多重比較検定によって評価し統計分析を行った。
これらの結果から近位尿細管のミトコンドリア損傷において、DF処置マウスの尿細管の細胞質では COX IV陽性顆粒数の増加が認められ、また、6時間群と比較して12および24時間群の変性した近位尿細管において、陽性顆粒の増加がより顕著であった。さらに、免疫染色スコアは、対照群と比較して、12および24時間群で有意に増加し、COX IVおよび 8-nitroguanosine陽性顆粒は、変性した近位尿細管細胞の細胞質内のほぼ同じ位置での局在が認められた。一方、近位尿細管におけるオートファジーの確認のためのp62の免疫組織化学的解析では、12および24時間群の変性近位尿細管に多数の陽性顆粒の存在が確認され、12時間群で最大値を示した。p62と 同様にLC3においても、24時間群よりも12時間群でより多くの陽性細胞が観察され、LC3陽性顆粒と LAMP-1陽性顆粒の共局在が認められた。
ウェスタンブロッティング
凍結した腎皮質を用い、LC3-I（16 kDa）および LC3-II（14 kDa）についてウェスタンブロット解析を行った。この検出結果をImage JおよびPhotoshopを用いて半定量解析を行った。対照群の発現レベルと比較して、12および24時間群におけるLC3-I発現の有意な増加が認められた。
近位尿細管の電子顕微鏡所見
腎皮質の電子顕微鏡所見において、DF処置群では対照群と比較し、より多くのミトコンドリアの変性、膨化および断片化が認められた。 6時間群では軽度のミトコンドリア変性が観察されたが、12および 24 時間群では重度のミトコンドリア変性が顕著に認められた。 また、12および24時間群ではほとんどの変性ミトコンドリアが断片化していることも確認された。 変性ミトコンドリアを含むオートファゴソームは6時間群では認められず、12時間群で顕著に認められた。さらに24時間群においては、オートファゴソーム像は減少し、分断化して変性したミトコンドリアが多数認められた。
NSAIDsは、アラキドン酸カスケードにおけるシクロオキシゲナーゼの阻害を通じて腎臓の血行動態を変化させ、刷子縁の喪失、尿細管の拡張、進行性病変の形成などの腎損傷を示す腎構造の変化を引き起こすことによりAKIを誘発するとされており、中でもDFはミトコンドリアを標的とし、ROS産生を介して腎毒性を誘発し、酸化ストレスと重大な DNA断片化を引き起こすことが報告されている。近年の報告では、ミトコンドリア膜電位、マイトファジーおよびミトコンドリアの断片化について培養細胞や腎組織の皮質全体を材料として検討されているが、本研究ではDFによる 損傷のターゲットの1つである腎臓の近位尿細管に焦点を当てて病理学的に検討した。DF誘発による酸化ストレスが DNA損傷を引き起こし、損傷した DNAの断片は細胞小器官、核またはミトコンドリアDNAのニトロ化核酸塩基として局在する可能性があり、免疫組織化学的にCOX IV陽性の細胞質顆粒は、損傷したミトコンドリアDNAを示すものと解釈された。COX IVと8-nitroguanosine免疫組織化学分析により、DF処置マウスの変性した近位尿細管細胞におけるこれらの局在がほぼ一致していることが明らかになった。 また、オートファジー関連抗原（p62、LC3、LAMP-1）における免疫組織化学分析によりオートファジー活性はDF処置後12時間で最大を示すことが明らかとなった。加えて電子顕微鏡所見により、DF処理後 12時間で損傷したミトコンドリアを処理するオートファジー（マイトファジー）像が顕著に示され、24時間でマイトファジー像の減少が認められ、代わりに多くの損傷および断片化したミトコンドリアが観察された。この結果は免疫組織化学分析の結果と一致した。 一方、LC3-IおよびⅡのウェスタンブロッティングの結果では、オートファジー活性は24時間で最も高かった。このことについては冒頭でも述べたが、ウェスタンブロッティングのサンプルを採材する際、遠位尿細管や糸球体などの近位尿細管以外の組織が含まれることに起因している。今回、腎皮質の近位尿細管に焦点を絞り、解析したことにより近位尿細管において24時間よりも早い段階での傷害が明らかとなった。DFの毒性が、損傷したミトコンドリアを除去するオートファジー機構であるマイトファジーの活性化とともに、近位尿細管におけるミトコンドリアの損傷および断片化を引き起こすことが確認され、 さらに、近位尿細管におけるマイトファジー活性は、12時間と比較して24時間で低下しており、損傷したミトコンドリアの処理が停滞することで、マイトファジーのクリアランス能力を超えた損傷ミトコンドリアの蓄積により尿細管上皮細胞のさらなる損傷を引き起こすことが示唆された。
近年、ミトコンドリア機能不全に関する研究は、AKIの治療法を特定するための刺激的な新分野として浮上している。中でも、ミトコンドリアの断片化の抑制がアポトーシスの抑制につながること、ミトコンドリア分裂因子であるmitochondrial fission factorの阻害が虚血性腎損傷モデルマウスにおけるミトコンドリアの恒常性を維持すること、また、腎虚血プレコンディショニング（IPC）中にマイトファジーが活性化される場合、Fundc1がミトコンドリア分裂の調節に関与するなどの報告があり、AKIにおけるミトコンドリアの断片化は重要な役割を果たす。今回の超微形態学的所見においても12 および24時間のDF処置群の近位尿細管の細胞質に断片化したミトコンドリアが認められた。 この研究は、DF誘発性腎損傷を保護または改善するために近位尿細管ミトコンドリアを標的とし解析するための報告として非常に重要である。
多くの研究がDFによる処置後24時間で腎障害の発生を報告している。本研究においても血清学的分析およびウェスタンブロッティング解析の結果ではDF処置後24時間まではDF誘発性腎損傷の時間依存性の増悪を示した。これは近位尿細管以外の尿細管を含む腎皮質全体の損傷を反映していると考えられた。一方で今回、病理学的に近位尿細管に焦点を当てた結果、組織学的データの統計解析および電子顕微鏡所見による明確な裏づけにより、オートファジー活性がDF処置後12時間でピークに達することが確認された。同時に、DF処置後24時間では、オートファジー活性の低下および電子顕微鏡所見から多数の除去されていない損傷ミトコンドリアおよび断片化が認められた。p62、LC3およびLAMP-1が12時間で最大値を示したことから、24時間より早い段階の12時間においてオートファジー活性が高いことが明らかとなった。本研究により腎皮質全体の傷害は近位尿細管におけるマイトファジーの停滞すなわち損傷ミトコンドリア蓄積により悪化する可能性があり、今後のAKIの予防への応用として、初期段階で近位尿細管のミトコンドリアの恒常性を維持することでDF誘発性のAKIを軽減できる可能性があることが示唆された。doctoral thesi
Impact of maternal fructose intake on liver stem/progenitor cells in offspring: Insights into developmental origins of health and disease
Get PDFAims: The developmental origin of health and disease (DOHaD) theory postulates that poor nutrition during fetal life increases the risk of disease later in life. Excessive fructose intake has been associated with obesity, diabetes, and nonalcoholic fatty liver disease, and maternal fructose intake during pregnancy has been shown to affect offspring health. In this study, we investigated the effects of high maternal fructose intake on the liver stem/progenitor cells of offspring.
Main method: A fructose-based DOHaD model was established using Sprague-Dawley rats. Small hepatocytes (SHs), which play an important role in liver development and regeneration, were isolated from the offspring of dams that were fed a high-fructose corn syrup (HFCS) diet. The gene expression and DNA methylation patterns were analyzed on postnatal day (PD) 21 and 60.
Key findings: Maternal HFCS intake did not affect body weight or caloric intake, but differences in gene expression and DNA methylation patterns were observed in the SHs of offspring. Functional analysis revealed an association between metabolic processes and ion transport.
Significance: These results suggest that maternal fructose intake affects DNA methylation and gene expression in the liver stem/progenitor cells of offspring. Furthermore, the prolonged retention of these changes in gene expression and DNA methylation in adulthood (PD 60) suggests that maternal fructose intake may exert lifelong effects. These findings provide insights into the DOHaD for liver-related disorders and highlight the importance of maternal nutrition for the health of the next generation.journal articl
Effect of dietary therapy on the postprandial serum lipid concentrations in dogs
Get PDFSerum triglyceride (TG) and Total Cholesterol (T-Cho) concentrations have been known to elevate postprandially. The objective of this study was to evaluate the effect of dietary therapy on the postprandial change of serum TG and T-Cho concentrations in dogs. Healthy 6 Beagles were randomly divided into 3 groups, and fed with high-energy diet, low-fat diet, or high-fiber diet for 2 weeks under 3×3 Latin Square protocol. Baseline and postprandial (2, 4, 6, 8, 10, and 12 hours after feeding) serum TG and T-Cho concentrations were measured and compared among the 3 dietary therapies. Only high-energy diet induced significant elevation of serum TG concentration postprandially. Serum TG concentrations 2–10 hours after feeding with high-energy diet were significantly higher than those with other 2 diets. Serum TG concentration was returned within reference range 12 hours after feeding with each diet. Serum T-Cho concentration did not show postprandial elevation. High-energy diet provided with a tendency to elevate the baseline serum T-Cho concentration when compared with other 2 diets, but there was no statistically significant difference. Although the baseline concentrations of TG and T-Cho was equivalent, high-energy diet could induce potential hypertriglyceridemia due to the dairy postprandial elevation of serum TG concentration. The effect of dietary therapy on the dogs with hyperlipidemia needs to be investigated.高脂血症は血清中のトリグリセリド（TG）および総コレステロール（T-Cho）濃度に基づいて診断されており、これらは食後に上昇することが知られている。本研究では、食事療法の違いによる食後の血清TG・T-Cho濃度への影響を明らかにすることを目的とした。健常なビーグル6頭を2頭ずつ無作為に3群へ振り分け、3×3のラテン方格法により高栄養食、低脂肪食、または高繊維食を2週間ずつ給与した。各食事を2週間給与した時点の朝における食前および食後2、4、6、8、10、12時間における血清TGおよびT-Cho濃度を測定し、各食事間で比較検討した。血清TG濃度は、高栄養食でのみ有意な食後の上昇が認められ、低脂肪食および高繊維食と比べて食後2〜10時間において有意な高値を示した。一方で、いずれの食事でも食後12時間までに基準範囲内の水準にまで低下した。血清T-Cho濃度は、いずれの食事でも食後の上昇は認められず、全ての時間帯において食事の差異による有意差も認められなかった。高栄養食では血清TG濃度を長時間にわたって上昇させることから、日常的な高栄養食の給与によって慢性的な高TG血症を引き起こす可能性が示唆された。journal articl
An analysis of Biodiversity Education in;UNESCO Associated Schools;Project Network
Get PDFjournal articl
The Ideal and Reality of Mudie’s Select Library: An Analysis of the Lists of Works in the Advertisements
Get PDFFor Mudie’s Select Library, a leading circulating library in the Victorian era, advertisements in magazines serve as a vital tool not only for acquiring new subscribers but also for maintaining and strengthening its ideal image. This study took lists of works from full-page advertisements in 1860, 1864, and 1873, and compared them with Mudie’s contemporaneous catalogs, which list fiction and nonfiction separately, in order to accurately categorize each piece and analyze the placement of fiction and nonfiction. Consequently, the list in 1860, when subscribers began to complain that there were no novels they wanted to read, appealed to people that Mudie’s was a select library. The 1864 list of works enhanced the presence of fiction, aiming to increase subscribers after transitioning to a limited liability company. Lastly, the 1873 list demonstrated an intention to shift its focus back to strengthening its ideal image after overcoming financial difficulties. In other words, the three lists of works emphasized alternately image (ideal) and management (reality), considering the trends of rival circulating libraries, Mudie’s business situation, the criticism directed towards the select library, and so forth.journal articl
牛伝染性リンパ腫ウイルスの血中遺伝子量と 乳房炎の免疫動態および経済的損失に関する研究
Get PDF麻布大学博士（獣医学）Bovine mastitis is a disease of great concern to dairy farmers due to its direct impact on milk production. It can be classified into clinical mastitis with clinical symptoms and subclinical mastitis without clinical symptoms, both of which can cause significant economic losses in the dairy industry. In Japan, treatment of mastitis primarily relies on antibiotic therapy. However, in recent years, there have been recommendations for the appropriate use and reduction of antibiotic usage from the perspectives of food safety, public health and antibiotic resistance.
Enzootic Bovine Leukosis (EBL) is caused by bovine leukemia virus (BLV), which is integrated into the animal’s genome as a provirus. It has been reported that BLV proviral load (PVL), that is, the number of provirus copies, is closely related to the progression of BLV infection and the development of EBL. The relationship between blood PVL and productivity of dairy cows including mastitis is becoming clearer in recent years. However, there is yet to be clarified, including the relationship between PVL and severity of clinical mastitis, the effect of BLV infection on the severity of clinical mastitis, the economic loss associated with the treatment of clinical mastitis, and intramammary immunity when clinical mastitis occurs. Furthermore, the efficacy of mastitis treatment in BLV-infected cows has not been determined. Mastitis is of great concern to dairy farmers. If we could clarify the impact and economic losses of BLV infection on mastitis, this research would contribute to control measures for both mastitis and BLV infection. In addition, while there is no effective treatment for BLV infection, it is possible to cure or improve symptoms by taking measures after mastitis occurs.
Thus, the purpose of this study is to clarify the effect of BLV infection on the severity of clinical mastitis, economic losses, intramammary immune dynamics at onset of clinical mastitis, and to investigate non-antibiotic methods to treat BLV-infected cows with subclinical mastitis.
In Chapter 1, BLV-infected cows with mild, moderate, and severe clinical mastitis were classified into BLV-positive cows with a PVL of ≥1,000 (copies/10 ng DNA) (H-PVL cows) and BLV-positive cows with a PVL of <1,000 (copies/10 ng DNA) (L-PVL cows). The classification was performed using PVL 1,000 (copies/10 ng DNA) as a diagnostic index suggested for predicting EBL, and the severity of clinical mastitis and innate immunity in milk at the onset of clinical mastitis were analyzed. BLV proviral load (PVL) showed significant correlation with the severity of clinical mastitis and lingual antimicrobial peptide concentration in milk. These results suggested that BLV-positive cows with a PVL of ≥1,000 (copies/10 ng DNA) are associated with higher severity of clinical mastitis compared to BLV-positive cows with a PVL of <1,000 (copies/10 ng DNA) due to decreased immune function of epithelial cells in the mammary gland.
In Chapter 2, an analysis was performed to establish a threshold value of PVL to identify an increased risk of severe clinical mastitis. Next, BLV-infected cows were classified based on the newly established PVL threshold value. Subsequently the prognosis and economic loss of clinical mastitis were evaluated based on this PVL value. Having defined the cutoff value of blood PVL as 17.8 (copies/10 ng DNA), the percentage of cows that were given systemic treatments and the technical fees for medical treatment were both significantly higher in BLV-positive cows above the PVL cut-off value than in BLV-positive cows below the PVL cut-off value and the negative group. Number of treatments was significantly higher in the group above the cut-off value than in the group below the cut-off value. There was no significant difference in prognosis of mastitis among the three groups. Therefore, BLV-positive cows above the PVL cut-off value may develop severe clinical mastitis but recover to remain in the herd with adequate and necessary treatment. However, it was suggested that there is a potential for significant expenses and time losses associated with medical treatment. The results of this study suggested that the PVL cut-off value of 17.8 (copies/10 ng DNA) is a useful threshold for increased economic losses in terms of increased costs and number of treatments for clinical mastitis in BLV-infected cows. Furthermore, this cut-off value may serve as a new standard value for the detection and culling of asymptomatic BLV-infected cows, particularly when considering the severity of clinical mastitis and the associated economic losses. This threshold may therefore be valuable for the prevention, treatment, and control of mastitis on farms in Japan.
In Chapter 3, using the PVL cut-off value of 17.8 (copies/10 ng DNA) established in Chapter 2, BLV-infected cows with subclinical mastitis were classified into BLV-positive cows above the PVL cut-off value and those below the PVL cut-off value. This chapter investigated the effect of Levamisole (LMS), known as an immunomodulator, on subclinical mastitis in BLV-infected cows. The bacterial count, somatic cell count and the number of CD172α+CD14+ positive cells (monocytes/macrophages) in milk of both BLV-negative cows and in BLV-positive cows below the PVL cut-off value were decreased by LMS administration. Therefore, it is suggested that LMS administration could improve the function of macrophages in milk and reduce bacterial and somatic cell count in BLV-negative cows and BLV-positive cows below the PVL cut-off value. Furthermore, the results indicated that a reduction in bacterial count in milk might be expected even in BLV-positive cows above the PVL cut-off value. It was also suggested that the PVL cut-off value of 17.8 (copies/10 ng DNA) established in Chapter 2 may serve as a criterion for assessing the efficacy of LMS administration in BLV-infected cows with subclinical mastitis.
The result of this study clarified the relationship between BLV infection and the severity of clinical mastitis, as well as the relationship between BLV infection and intramammary natural immunity factor at the onset of clinical mastitis. The PVL cut-off value of 17.8 (copies/10 ng DNA) was newly established as a threshold for increased risk of severe clinical mastitis and economic loss associated with treatment of clinical mastitis in BLV-infected cows. Furthermore, this study demonstrated the possibility of improving subclinical mastitis in BLV-positive cows through treatment with an immunomodulator.
In conclusion, the findings of this study are expected to raise awareness regarding the risks of mastitis associated with BLV infection among veterinarians involved in mastitis treatment, all staff engaged in BLV eradication, and dairy farmers. Furthermore, the findings of this study could be used towards a new approach to reduce economic losses due to mastitis in BLV-infected cows.牛の乳房炎は生乳生産に直接影響を与えることから、酪農家の関心が非常に高い疾病である。臨床症状を認める臨床型乳房炎と臨床症状を認めない潜在性乳房炎に大別でき、どちらも酪農経済に甚大な被害を及ぼす。日本における乳房炎の治療は抗菌剤による治療が大部分を占めているが、近年、食の安全・安心や薬剤耐性の観点から抗菌剤の適正使用・使用量の削減が提言されている。
地方病型牛伝染性リンパ腫（EBL）は、牛伝染性リンパ腫ウイルス（BLV）感染に起因し、感染したBLVはプロウイルスとして宿主ゲノムに組み込まれる。このプロウイルスコピー数を表すプロウイルスロード（PVL）は、病態の進行やEBL発症に深く関わっていることが知られている。BLV感染においては、近年、血中PVLと、乳房炎を始めとした生産病との関連が既に明らかになりつつあるが、PVLとの関連を含めBLV感染が臨床型乳房炎の重篤度や診療費用、乳房内の免疫状態に及ぼす影響、さらにBLV感染牛における乳房炎の治療や改善方法は明らかにされていない。酪農家の関心の高い乳房炎に対してBLV感染が及ぼす影響や経済損失を明らかにできれば、乳房炎とBLV感染の双方の制御対策に貢献できると考えられる。また、BLVは一度感染してしまうと有効な治療法は存在しないが、乳房炎は感染が成立した後でも処置を行うことで治癒や症状の改善を目指すことが可能である。
そこで本研究は、BLV感染が臨床型乳房炎の重篤度に与える影響や経済損失、臨床型乳房炎を発症した際の乳房内の免疫動態を明らかにすることを目的とし、さらに、抗菌剤を使用せずにBLV感染牛における潜在性乳房炎を改善する方法を検討した。
第1章では、軽度、中等度及び重度の臨床型乳房炎を発症したBLV感染牛を、EBL発症の診断基準PVL 1,000（copies/10 ng DNA）を用いて、1,000（copies/10 ng DNA）以上を示すBLV陽性牛（H-PVL群）と、未満を示すBLV陽性牛（L-PVL群）に分類し、臨床型乳房炎の重篤度と臨床型乳房炎発症時の乳汁中の自然免疫を解析した。解析により、血中のPVLは臨床型乳房炎の重篤度及び、乳汁中の自然免疫因子であるLingual antimicrobial peptide濃度に有意な関連を示したことから、血中PVL 1,000（copies/10 ng DNA）以上を示すBLV陽性牛では血中PVL 1,000（copies/10 ng DNA）未満のBLV陽性牛に比べて、乳房内の乳腺上皮細胞の免疫機能の低下により、臨床型乳房炎が重篤化することが推察された。
第2章では、BLV感染牛における臨床型乳房炎の重篤化リスクが増す血中PVLの指標設定を目的として解析を行い、新規に設定した血中PVLに基づいてBLV感染牛を分類し、臨床型乳房炎の予後と臨床型乳房炎治療に伴う経済損失を解析した。結果から、臨床型乳房炎の重篤化リスクが増す血中PVLのカットオフ値を17.8（copies/10 ng DNA）と設定したところ、血中PVLカットオフ値以上のBLV陽性牛では、カットオフ値未満及びBLV陰性牛に比較して、重篤な臨床型乳房炎の発生件数及び全身治療割合が多く、治療に伴う技術料も高額となり、診療回数もカットオフ値未満のBLV陽性牛に比較して多くなった。血中PVLカットオフ値に関わらず予後には差が見られなかったことから、血中PVLカットオフ値以上のBLV陽性牛は重篤な臨床型乳房炎を発症するものの、適切な治療を必要十分に行うことで牛群に残留できるまでに回復するが、診療にかかる経費や時間の損失は大きくなることが推察された。今回得られた血中PVLのカットオフ値17.8（copies/10 ng DNA）は、BLV感染牛における臨床型乳房炎の診療費および診療回数の増加という経済損失を増大させる指標として有用である可能性が示唆された。さらに、このカットオフ値は、臨床型乳房炎の重篤度およびそれに伴う経済的損失を考慮した場合における無症状BLV感染牛の新たな摘発淘汰基準値として、また、農場における乳房炎の防除対策や治療方針の指標として日本国内で活用できる可能性が示唆された。
第3章では、第2章で設定した血中PVLのカットオフ値 17.8（copies/10 ng DNA）を利用して、潜在性乳房炎を発症したBLV感染牛を血中PVLカットオフ値 17.8（copies/10 ng DNA）以上のBLV陽性牛と未満を示すBLV陽性牛に分類し、免疫賦活剤としても知られる塩酸レバミゾール（LMS）を用いて潜在性乳房炎に対する効果を検討した。LMS投与により、BLV陰性牛及び血中PVLカットオフ値未満のBLV陽性牛において、体細胞数および生菌数の減少という臨床的な効果が認められ、乳汁中マクロファージの機能が活性化されたことが推察された。ついで、血中PVLカットオフ値以上のBLV陽性牛であっても、生菌数の減少に対する効果が期待できることが明らかとなった。また、第2章で提案した血中PVLのカットオフ値17.8（copies/10 ng DNA）が、 BLV感染牛における免疫賦活剤を用いた潜在性乳房炎の改善効果の判定基準としても利用できることが示唆された。
本研究は、臨床型乳房炎の重篤度および臨床型乳房炎発症時の乳房内の自然免疫因子とBLVの病態との関連を明らかにし、血中PVL 17.8（copies/10 ng DNA）を臨床型乳房炎の重篤化リスクおよび臨床型乳房炎の治療に伴う経済損失が増加する指標として示すことができた。さらに、免疫賦活剤を用いることでBLV感染牛の潜在性乳房炎を改善できることを示した。
本研究で得られた知見は、乳房炎治療に関わる獣医師、BLV清浄化対策に関わる全ての関係者、そして酪農家に、BLV感染が乳房炎におよぼすリスクを再認識させ、BLV感染牛における乳房炎による経済損失を軽減するための新しいアプローチとして有用である。doctoral thesi