我们提出了一个方法,形成一个横跨毫米,是没有大脑的炎症个月稳定在小鼠颅骨的影像窗口。这种方法非常适合于使用双光子显微镜的血液流通,细胞动力学,细胞/血管结构的纵向研究。
在体内的皮质功能成像需要光纤接入无脑颅内环境的破坏。我们提出了一个方法,在小鼠颅骨,跨越直径几毫米个月的稳定形成抛光和钢筋颅骨变薄(端口)窗口。手举行演习,以实现光学清晰度头骨10至15微米的厚度变薄,然后覆盖氰基丙烯酸酯胶和玻璃盖:1)提供刚性,2)抑制骨的再生和3)减少光散射从骨表面上的违规行为。由于颅骨没有违反,任何可能会影响正在研究过程中的炎症,会大大减少。可以实现利用双光子激光扫描显微镜成像深度可达250微米以下的皮质表面。这个窗口非常适合研究脑血流和细胞的功能,在麻醉和清醒的准备。它进一步提供运portunity操纵细胞的活性,利用光遗传学或破坏由照射循环光敏剂的目标船只的血流量。
通过一个端口“窗口中的双光子成像需要通过传输薄骨和硬脑膜,衰减激光和光学像差增加了更大的深度8。然而,尽管这个缺点,成像深度可达250微米以下脑膜表面可以达到900 nm激发。大成像深度可能在原则上有可能与较长的激发波长13。这种方法的主要优点是皮质炎症的情况下,可能存在短暂的涉及全开颅14,15的方法。一个精心准备的端口窗口应显示血管生成或炎症没…
The authors have nothing to disclose.
这项工作是由美国心脏协会(博士后奖学金AYS)和国立卫生研究院(MH085499,EB003832,并OD006831 DK)的支持。我们感谢贝思·弗里德曼和巴勃罗·布林德手稿上的评论。
Agent | Route of delivery | Dose for mouse | Duration | Notes | Source | Ref Ref |
Pentobarbital (Nembutal) | IP | 90 μg/g | 15-60 min | Narrow safety margin. Work up to proper dose of anesthesia slowly. Supplement 10 % of induction dose as required. | 036093; Butler Schein | 7 |
Ketamine (Ketaset) mixed with Xylazine (Anased) | IP | 60 μg/g (K)
10 μg/g (X) (mix in same syringe) |
20-30 min | Xylazine is co-injected as a muscle relaxant and analgesic. Supplement only Ketamine at 50% of induction dose as required. | (K) 010177, (X) 033198; Butler Schein | 7 |
Isoflurane (Isothesia) | Inhalation | 4% mean alveolar concentration (MAC) for induction; 1-2% MAC for maintenance | 4-6 h. | Provided in mixture of 70% oxygen and 30% nitrous oxide. Prolonged anesthesia leads to slow recovery. | 029403; Butler Schein | 26 |
Table 1. Suggested anesthetic agents for survival studies.
ITEM | COMPANY | CATALOG # / MODEL |
Betadine | Butler Schein | 6906950 |
Buprenorphine (Buprenex) | Butler Schein | 031919 |
Fluorescein isothiocyanate dextran, 2 MDa molecular weight | Sigma | FD2000S |
Isopropyl alcohol | Fisher | AC42383-0010 |
Lactated Ringer’s Solution | Butler Schein | 009846; |
Lidocaine solution, 2 % (v/v) | Butler Schein | 002468 |
Saline | Butler Schein | 009861 |
Surgical Milk | Butler Schein | 014325 |
Texas Red dextran, 70 kDa molecular weight | Invitrogen | D1864 |
Maxizyme | Butler Schein | 035646 |
DISPOSABLES | ||
Carbide burrs, 1/2 mm tip size | Fine Science Tools | 19007-05 |
Cottoned tip applicators | Fisher Scientific | 23-400-100 |
Cover Glass, no. 0 thickness | Thomas Scientific | 6661B40 |
Cyanoacrylate glue | ND Industries | 31428 H04308 |
Gas duster | Newegg | N82E16848043429 |
Grip cement powder | Dentsply | 675571 |
Grip cement solvent | Dentsply | 675572 |
Insulin syringe, 0.3 mL volume with 29.5 gauge needle | Butler Schein | 018384 |
Nut and bolt to secure the head | Digikey | Nut, H723-ND; bolt, R2-56X1/4-ND |
Opthalmic ointment | Butler Schein | 039886 |
Scalpel blades | Fisher Scientific | 12-460-448 |
Screws, self-tapping #000 | J.I. Morris Company | FF000CE125 |
Silicone aquarium sealant | Perfecto Manufacturing | 31001 |
Tin oxide powder | Mama’s Minerals | EQT-TINOX |
EQUIPMENT | ||
Glass scribe | Fisher Scientific | 08-675 |
Dissecting microscope | Carl Zeiss | OPMI-1 FC |
Electric powered drill | Foredom or Osada | K.1020 (Foredom) or EXL-M40 (Osada) |
Electrical razor | Wahl | Series 8900 |
Forceps, Dumont no. 55 | Fine Science Tools | 11255-20 |
Feedback regulated heat pad | FHC | 40-90-8 (rectal thermistor, 40-90-5D-02; heat pad, 40-90-2-07) |
Isoflurane vaporizer | Ohmeda | IsoTec4 |
Pulse oximeter | Starr Life Sciences | MouseOx |
Screwdriver, miniature | Garret Wade | 26B09.01 |
Stereotaxic frame | Kopf Instruments | Model 900 (with mouse anesthesia mask and non-rupture ear bars) |
Surgical scissors, blunt end | Fine Science Tools | 14078-10 |
Ultrasonic cleaner | Fisher Scientific | 15-335-30 |
Table 2. List of specific reagents, disposables and equipment.