渗透压微型泵植入增加小鼠脑脊液中葡萄糖浓度
Summary
本文描述了增加小鼠脑脊液（CSF）中葡萄糖浓度的详细方案。这种方法可用于研究高脑脊液葡萄糖对小鼠神经变性、认知和外周葡萄糖代谢的影响。
Abstract
糖尿病会增加认知能力下降的风险并损害大脑功能。高血糖和认知缺陷之间的这种关系是否是因果关系仍然难以捉摸。此外，这些缺陷是否由脑脊液（CSF）和/或血液中葡萄糖水平升高介导也不清楚。很少有研究调查高脑脊液葡萄糖水平对中枢神经系统（CNS）功能的直接影响，特别是对学习和记忆的影响，因为目前的糖尿病模型还不足以解决此类研究问题。本文介绍了一种通过使用渗透微泵将葡萄糖连续注入小鼠侧脑室来长期增加脑脊液葡萄糖水平 4 周的方法。该方案通过测量脑脊液中的葡萄糖水平进行了验证。该方案在以0.25μL / h流速输注50%葡萄糖溶液后将CSF葡萄糖水平提高至~328mg / dL，而接受人工脑脊液（aCSF）的小鼠的CSF葡萄糖浓度为~56mg / dL。此外，该方案不影响血糖水平。因此，该方法可用于确定高脑脊液葡萄糖对脑功能或特定神经通路的直接影响，而与血糖水平的变化无关。总体而言，这里描述的方法将有助于开发动物模型，用于测试高脑脊液葡萄糖在介导阿尔茨海默病和/或其他与糖尿病相关的神经退行性疾病特征中的作用。
Introduction
1型和2型糖尿病都会损害大脑功能1，2，3。例如，糖尿病会增加认知能力下降和神经退行性疾病的风险，包括阿尔茨海默病3，4。此外，糖尿病患者的大脑葡萄糖感应有缺陷5，6。这种缺陷导致低血糖相关的无意识和对低血糖7，8的反调节反应不足的发病机制，如果不立即治疗，这可能是致命的。
考虑到糖尿病会增加血液和脑脊液（CSF）中的葡萄糖水平9，重要的是要确定这些因素中的一个或两个是否会导致脑功能受损。糖尿病是否单独通过高脑脊液葡萄糖或与其他因素（如胰岛素缺乏或胰岛素抵抗）相结合导致脑损伤也是一个悬而未决的问题。1型和2型糖尿病的动物模型除了受影响的能量平衡和外周葡萄糖代谢外，还显示认知能力下降和神经变性10，11，12，13。然而，从这些模型中，将高脑脊液葡萄糖与血糖水平在介导糖尿病并发症对脑功能中的选择性作用解耦是不可行的。
该协议描述了开发高血糖症小鼠模型的方法，以测试长期高脑脊液葡萄糖水平对脑功能，能量平衡和葡萄糖稳态的影响。通过该技术开发的小鼠模型为研究失调的葡萄糖稳态对神经和行为功能的病因学作用提供了一种工具。
因此，所提出的方法将有助于理解脑脊液葡萄糖水平升高在各种病理生理条件下的直接影响。
Protocol
所有小鼠程序均由罗切斯特大学机构动物护理和使用委员会批准，并根据美国公共卫生服务关于实验动物人道护理和使用指南进行。用于本研究的六周龄C57BL / 6J雄性小鼠是商业获得的。所有动物都被分组饲养（每个笼子5只小鼠）在一个昼夜循环12小时的房间里，并随意获得食物 和水。在将小鼠植入套管以将葡萄糖注入侧脑室后，将它们单独容纳以防止其他小鼠对植入物的任何损害。 <p …
Representative Results
将雄性小鼠植入组装到渗透微型泵上的套管（图1），以将aCSF或50%葡萄糖溶液长期注入其侧脑室（图2）。在手术后10天收集脑脊液（图3）以验证该手术的有效性。结果显示，与输注aCSF的小鼠（平均值：56.5mg / dL）相比，输注50%葡萄糖的小鼠的CSF葡萄糖水平（平均值：327.7mg / dL）增加。与对照同窝小鼠相比，实验小鼠的脑脊液葡萄?…
Discussion
本文报告了通过使用连接到植入侧脑室的套管的渗透微型泵来增加小鼠脑脊液葡萄糖的详细方案。通过该程序在小鼠大脑中长期输注葡萄糖将有助于描述长期高血糖对认知，全身葡萄糖代谢和能量平衡的影响，并有助于更好地了解糖尿病并发症的发病机制。
慢性糖尿病会导致脑损伤，中断大脑和周围器官之间的交流15。糖尿病还会增加神经退行性疾病的风险…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
美国国立卫生研究院向KHC拨款DK124619。
纽约州罗切斯特大学医学系的启动资金和试点研究奖授予KHC。
罗切斯特大学德尔蒙特神经科学研究所试点研究奖授予KHC。
纽约州罗切斯特大学研究副校长办公室大学研究奖授予KHC。
MUR设计并执行了该方法，分析了结果，准备了图形和数字，并撰写和编辑了手稿。KHC构思并监督了这项研究，分析了结果，并撰写和编辑了手稿。KHC是这项工作的担保人。所有作者都批准了手稿的最终版本。
Materials
| 0.22 µm syringe filter | Membrane solutions | SFPES030022S | |
| 1 mL sterile Syringe (Luer-lok tip) | BD | 309628 | |
| 1 mL TB syringe | BD | 309659 | |
| 100 mL Glass beaker | Fisher | N/a | |
| 100% Ethanol (Koptec) | DLI | UN170 | Use 70% dilution to clean the surgery area |
| 50 mL conical tube | Fisher | N/A | |
| Allignment indicator | KOPF | 1905 | |
| Alzet brain infusion kit | DURECT | Kit # 3; 0008851 | Cut tubing in the kit to 1 inch length |
| Alzet osmotic pump | DURECT | 2004 | Flow rate 0.25 µL/h |
| Anesthesia system | Kent Scientific | SomnoSuite | |
| Betadine solution | Avrio Health | N/A | |
| CaCl2 . 2H2O | Fisher | C79-500 | |
| Cannula holder | KOPF | 1966 | |
| Centering scope | KOPF | 1915 | |
| Dental Cement Liquid | Lang Dental | REF1404 | |
| Dental cement Powder | Lang Dental | REF1220-C | |
| D-glucose | Sigma | G8270 | |
| Electric drill | KOPF | 1911 | While drilling a hole avoid rupturing dura mater |
| Eye lubricant (Optixcare) | CLC Medica | N/A | |
| Glass Bead sterilizer (Germinator 500) | VWR | 101326-488 | Place instruments in sterile water to let them cool before surgery |
| Glucose Assay Kit | Cayman chemical | 10009582 | |
| H2O2 | Sigma | H1009-500ml | Apply 3% H2O2 on skull surface to make the cranial sutures visible. |
| Hair Clipper | WAHL | N/A | |
| heating pad | Heatpax | 19520483 | |
| Hemostat | N/A | N/A | |
| Isoflurane (Fluriso) | Zoetis | NDC1385-046-60 | |
| KCl | VWR | 0395-500g | |
| Magnetic stand | WPI | M1 | |
| Magnifying desk lamp | Brightech | LightView Pro Flex 2 | |
| Metal Spatula | N/A | N/A | |
| MgCl2 . 6H2O | Fisher | BP214-500 | |
| Micromanipulator (Right handed) | WPI | M3301R | |
| Micromanipulator with digital display | KOPF | 1940 | |
| Na2HPO4 . 7H2O | Fisher | S373-500 | |
| NaCl | Sigma | S7653-5Kg | |
| NaH2PO4 . H2O | Fisher | S369-500 | |
| Neosporin | Johnson & Johnson | N/A | Apply topical oinment to prevent infection |
| Parafilm | Bemis | DM-999 | |
| Rimadyl (Carprofen) 50mg/ml | Zoetis | N/A | 5 mg/kg, subcutaneous, for analgesia |
| Scalpel | N/A | N/A | |
| Stereotaxic allignment system | KOPF | 1900 | |
| Sterile 27 gauge needle | BD | 305109 | |
| Sterile cotton tip applicators (Solon) | AMD Medicom | 56200 | |
| Sterile nylon sutures (5.0) | Oasis | MV-661 | Use non-absorable suture for closing the wound |
| Sterile sharp scissors | N/A | N/A | |
| Sterile surgical blades | VWR | 55411-050 | |
| Surgical gloves (Nitrile) | Ammex | N/A | Change gloves if there is suspision of contamination |
| Tray | N/A | N/A |
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Diesen Artikel zitieren
Raza, M. U., Chhabra, K. H. Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid. J. Vis. Exp. (194), e65169, doi:10.3791/65169 (2023).