アルツハイマー病動物モデルを用いた痴呆の定量化と抗痴呆療法の研究

金沢大学医学部・附属病院アルツハイマー病モデルとしての前脳基底核(NBM)破壊ラットに対して自家頸部迷走神経節(以後,X)を脳内移植後,4週間経時的にアセチルコリン(ACh)系マーカー(choline acetyltransferase:CAT,acetylcholine transesterase:AChE活性,^3H-QNB結合能測定によるムスカリン受容体(mAChR)と事象関連電位(P300)を連続測定し,X移植の治療効果を判定した.NBM破壊とX移植の確認のためAChE染色(Tago法)を行った.その結果,移植Xは4週後も脳内に生着し,AChE染色に陽性であった.P300潜時は正常群で356.6±21.3msec.破壊群では破壊4週後も400msec以上に遅延したが,X移植群では移植後2週目から351.0〜360.0msec以下に改善した.ACh系マーカーは,破壊群で1週後,破壊側CAT活性が健側に比べCC部で約65%に,SS部で約75%に低下した.破壊2週以後はSS部CAT活性は改善傾向を示したが,CC部は改善不良であった.3週後,AChE活性は最も低下した(CC部で著明)が,mAChR値はピークを示した.X移植治療群ではCAT活性は移植2週以後,CC,SS部ともに上昇改善した.AChE活性もSS部で2週以後,CC部で3週以後改善した.しかしmAChR値は移植2週以後,全脳において低下し続けた.感覚刺激に対する大脳皮質の電気的反応は新皮質表面から放出されるAChにより大きな調節を受けており,新皮質に投射するAChニューロンの集合体であるNBMの傷害は新皮質のCAT活性を低下させ,事象関連電位(P300)を抑制する.本実験でNBMの破壊後CAT活性が低下しP300の潜時が遅延したが,X移植後改善した.これは脳内に生着したXのAChニューロンがAChを産生したためと考えられた.NBM破壊3週後,一過性にAChE活性が低下しmAChR値が増加した現象は,CC部での急激なCAT活性低下によるACh減少に対する代償的変化と考えられた.これらはACh量によるAChEの産生調節,中隔野破壊後の海馬におけるムスカリン結合部位の増加とよく一致する.X移植後のAChEの漸増とmAChR値の漸減も生着したX内AChニューロンによるACh産生増加に並行した変化であると考えると説明し易い.本実験で以下の結論を得た.1.急激なCAT活性低下によるAChの減少に対し,AChE(ACh分解酵素)活性の低下と受容体結合能の増加という代償機構が働いた.2.アルツハイマー病モデルラットでXの脳内移植はCAT活性を高めコリン作動性の神経情報伝達を正常化した.A unilateral lesion was made in the nucleus basalis magnocellularis (NBM) of the Alzheimer rat model and after one week autotransplantation of the vagal nodosal ganglion (X) was carried out. Four weeks later serial measurements of Acetylcholine (Ach) markers (choline acetyltransferase, CAT ; acetylcholineesterase, AChE ; ^3H-QNB binding activity to muscarine receptor, mAChR) and latency of the P300 (event-related potentials) were carried out for the evaluation of its therapeutic effects. Histological studies by the AChE staining (Tago\u27s method) showed the NBM lesions made by microinjection of the ibotenic acid. Accepted and proliferated viable X tissue was documented in the brain even 4 weeks after X autoplantation. The latency of P300 was 356(〕SY.+-.〔)21.3 msec in normal rats. It was continuously delayd over 400 msec until 4 weeks in the NBM-lesioned rats. Whereas it was found less than 351.0-360.0 msec after 2 weeks in the autotransplanted rats ; indicating significant improvement. The changes of ACh markers was as follows : 1) One weeks after making lesion, CAT activity was reduced to approximately 65% in the cerebral cortex (CC) and 75% in the subcortical structure (SS) on the lesion side compared to that on the contralateral side. From 2 weeks, CAT activity was increased in CC but not in SS.After 3 weeks, AChE activity was reduced to the lowest level and on the contrary mAChR was increased to reach its peak. 2) CAT activity was increased in the CC and SS from 2 weeks after autotransplantation. AChE activity was also increased in SS from 2 weeks and in CC from 3 weeks. However, mAChR was reduced continuously from 2 weeks in the whole brain. From results mentioned above, the conclusions is as follows : 1) In response to the rapidly decreased ACh production caused by cholinergic deprivation, a kind of compensatory mechanism, such as the increased receptor-binding activity combined with the reduced AChE activity, might take place. 2) Autotransplantation of研究課題/領域番号:03670678, 研究期間(年度):1991 – 1993出典：研究課題「アルツハイマー病動物モデルを用いた痴呆の定量化と抗痴呆療法の研究」課題番号03670678（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-03670678/036706781993kenkyu_seika_hokoku_gaiyo/）を加工して作

脳血流再開後の二次的脳損傷に対する抗細胞間接着分子モノクローナル抗体療法

金沢大学医学部・付属病院家兎モデルを用いて脳虚血後再潅流障害における抗L-セレクチン中和モノクローナル抗体のE-セレクチン発現に対する治療効果を検索した。経眼窩的にウサギ内頚、中大脳、前大脳動脈をクリップして一過性閉塞(2.5時間)した後、3、6時間後にクリップを外して脳血流再潅流を行なった。このモデルに対して再潅流直後、2mgの抗Lセレクチン中和抗体(治療群)、または生理食塩水を静脈投与した(未治療群)。これら2群につき、免疫染色法によりEセレクチン(Endothelial Leukocyte Adhesion Molecule : ELAM-1)の発現部位とその程度を組織学的に検索した。そしてウサギELAM-1のオリゴヌクレオチド・プライマーをもとにRT-PCRでクローニングして得た一本鎖DNAをプローブとしてnorthern blottingを行ない、ELAM-1mRNAの発現状態を検索した。その結果、1)免疫組織学的には、ELAM-1は再潅流3時間後に梗塞巣を中心に最も強く発現し、6時間後には減弱した。ELAM-1の染色は梗塞巣を中心に周囲組織内の細動静脈および星状細胞ならびに神経細胞胞体に認められた。2)抗Lセレクチン中和抗体の投与によりELAM-1に対する染色範囲の縮小と染色性の低下を認めた。3)虚血脳においてELAM-1mRNAの発現に再潅流3時間後と6時間後とで差異はなく、抗Lセレクチン中和抗体を投与したものでも同様であった。以上より、脳虚血後再潅流障害早期に起こる血管内皮側の反応としてのEセレクチンのmRNAのupregulationに白血球側のLセレクチンは関係せず、炎症性サイトカインの刺激により誘導産生されたEセレクチンは内皮細胞と星状細胞、神経細胞内にすでに貯蔵されていてLセレクチンを介した刺激により細胞外へ放出されると考えられた。The effects of the anti-L selectin antibody on expression of E selectin were studied in a rabbit model of post-ischemic brain reperfusion. Anesthetized rabbits underwent 2.5 hours occlusion of the internal carotid, middle cerebral, and anterior cerebral artery with the trans-orbital approach, followed by 3 and 6 hours reperfusion. Just after the start of reperfusion, they were treated with either i.v.2 mg of anti-L selectin neutralizing antibody (treated group) or normal saline (untreated group). For these 2 groups, 1)the immunohisto-pathological study was carried out for evaluating the localization of the expressed E-selectin (Endothelial Leukocyte Adhesion Molecule : ELAM-1), and 2)The northern blotting was done for evaluating the expression of ELAM-1 mRNA with the single strand DNA probe of ELAM-1 by RT-PCR cloning using the oligonucleotide primer.Immmunohistopathologically, ELAM-1 expressed more strongly in the infarct area and its neighbor 3 hours after reperfusion than 6 hours. And it expressed not only in the small vessels but also in the cytoplasm of astrocytes and neurons around the infarct area. The administration of the neutralizing anti-L selectin antibody weakend the immunoreactivity to ELAM-1 in the 3 hours reperfusion model. Northern blotting showed no difference in expression of ELAM-1 mRNA between the treated group and the untreated one. In conclusion, L-selectin might play no role in upregulation of the ELAM-1 mRNA expression in the early stage of post-ischemic reperfusion brain injury. However, it might stimulate the release of ELAM-1, which is induced in the endothelial cells, astrocytes, and neurons by inflammatory cytokines and stored in them, to the extracellular space.研究課題/領域番号:06671379, 研究期間(年度):1994 – 1995出典：研究課題「脳血流再開後の二次的脳損傷に対する抗細胞間接着分子モノクローナル抗体療法」課題番号06671379（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-06671379/066713791995kenkyu_seika_hokoku_gaiyo/）を加工して作

発症早期ALS患者に対する超高用量メチルコバラミンの有効性・安全性について : ランダム化比較試験

Importance: Post hoc analysis in a phase 2/3 trial indicated ultra-high dose methylcobalamin slowed decline of the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) total score at week 16 as well as at week 182, without increase of adverse events, in patients with amyotrophic lateral sclerosis (ALS) who were enrolled within 1 year from onset. Objective: To validate the efficacy and safety of ultra-high dose methylcobalamin for patients with ALS enrolled within 1 year of onset. Design: A multicenter, placebo-controlled, double-blind, randomized phase 3 trial with 12-week observation and 16-week randomized period, conducted from October 2017 to September 2019. Setting: Twenty-five neurology centers in Japan. Participants: Patients with ALS diagnosed within 1 year of onset by the updated Awaji criteria were initially enrolled. Of those, patients fulfilling the following criteria after 12-week observation were eligible for randomization: 1- or 2-point decrease in ALSFRS-R total score, a percent forced vital capacity over 60%, no history of noninvasive respiratory support and tracheostomy, and being ambulant. The target number was 64 in both methylcobalamin and placebo groups. Of 203 patients enrolled in the observation, 130 patients (age, 61.0 ± 11.7 years; female, 56) met the criteria and were randomly assigned through an electronic web-response system to methylcobalamin or placebo (65 for each). Of these, 129 patients were eligible for the full analysis set, and 126 completed the double-blind stage. Interventions: Intramuscular injection of methylcobalamin 50 mg or placebo twice weekly for 16 weeks. Main outcomes and measures: The primary endpoint was change in ALSFRS-R total score from baseline to week 16 in the full analysis set. Results: The least-squares mean difference in ALSFRS-R total score at week 16 of the randomized period was 1.97 points greater with methylcobalamin than placebo (−2.66 versus −4.63; 95% CI, 0.44–3.50; P = 0.012). The incidence of adverse events was similar between the two groups. Conclusions and relevance: Ultra-high dose methylcobalamin was efficacious in slowing functional decline and safe in the 16-week treatment period in ALS patients in the early stage and with moderate progression rate. Trial registration: UMIN-CTR Identifier: UMIN000029588 (umin.ac.jp/ctr); ClinicalTrials.gov Identifier: NCT03548311 (clinicaltrials.gov

Retrocerebellar glio-ependymal cyst with corpora amylacea in wall. Case report

金沢大学医薬保健研究域医学系金沢大学附属病院脳神経外科The capsule of a retrocerebellar glio-ependymal cyst, which was incidentally found in a 54-year-old asymptomatic female, was studied by both light and electron microscopy. Microscopically, the capsule measured 25-400 ﾎｼm in thickness and consisted of four layers. The external covering was an arachnoid cell layer abutting on the thick connective tissue with scattered vessels, whereas the luminal lining was a layer of ependymal cells abutting on the glial tissue. The ependymal cells were cuboidal in shape and some, but not all, were ciliated. The glial tissue underneath the ependymal lining contained numerous corpora amylacea, especially in its abluminal side. Electronmicroscopically, the arachnoid cell layer consisted of both arachnoid epithelium and the underlying arachnoid cells. The former showed a relatively higher electron density than the latter. There was a distinct basement membrane between them. In the former, a moderate number of desmosomes and intermediate junctions assured contacts between the arachnoid cells. In the latter, there were many extracellular clefts containing numerous collagen fibrils and microfibrils. Neighboring cytoplasmic processes often formed interdigitations. The luminal surface of ependymal cells displayed variable numbers of microvilli with or without cilia, whereas the abluminal surface abutted directly on the astrocytic processes of the glial layer. There were some interdigitations of the plasmalemma of adjacent cells, with surface specializations consisting of fasciae adherentes joined by gap junctions. Two or more adjacent cells contributed to the development of microrosettes, packed with both numerous surface microvilli and several profiles of cilia. The corpora amylacea varied 5-15 ﾎｼm in diameter and consisted of randomly oriented filaments with the fine osmiophilic granules. The fibrillar elements within the processes of the fibrillary astrocytes swept around the corpora amylacea or impinged upon the latter. Although their outline was usually smooth and clearly demarcated, no membrane or space intervened between the corpora amylacea and the surrounding structures. It is most probable that the present glio-ependymal cyst arose from the displaced segment of the wall of the neural tube, which corresponds to the sites from which the tela chorioidea forms