Friday, June 8, 2007

Intraperitoneal Injection (IP) --- 腹腔注射

IP是一个很普通的注射方式。哺乳类动物的腹部,有所谓的腹腔(peritoneal cavity)。腹腔里面有腹部的器官,和血管等。腹腔由一层腹腔膜所覆盖。所以我们肚皮的下面,还有一层腹腔膜,腹腔膜里面才是腹部器官。

要进行IP,一样也要先替老鼠量体重,计算剂量。一般我都是打0.5ml的液体进入老鼠腹腔,小鼠(mouse)腹腔不大,太多的液体不太适合,所以习惯了打0.5ml。剂量就要根据这个来计算。

举例,你有一瓶毒素,剂量为10mg/ml。而你打算给老鼠打的剂量是5ug/g in 0.5ml:
*ug是microgram,比mg少1000倍*

Drug Stock: 10mg/ml
Desired dosage: 5ug/g

For example, a mouse weighs 25g,
for 1g of mouse inject 5ug of drug,
hence 5ug x 25g=125ug for a 25g mouse.

To inject 0.5ml into the mouse,
you have to have a concentration of 250ug/ml of your drug.
1ml contains 250ug, hence 0.5ml contains 125ug.

Dilution from the drug stock of 10mg/ml to 250ug/ml: 1:40 times dilution.
For example, if you want to prepare 2ml of 250ug/ml, then add 50ul of the stock to 1950ul of buffer.

像上面这种算术,是生物学要求的最基本数学,也是最常用的算术。

算好了剂量,做好了稀释(dilution)之后,就开始要load进针筒里。一般我打IP都用26G的针头。针头的大小(diameter)跟gauge(G)的数目成反比,即号码越小,针头越大。26G蛮细的,适合打小鼠的肚子。针筒里最好不要有泡泡,不然volume不准确,还有打IV的话你是不要把气泡打进人家的血管里面的罗。

要弄掉针筒里的泡泡,我一般的做法是,先提取了一定volume的液体,然后倒反针筒,往后拉plunger,拉到上面出现很多空位,再慢慢从后面推plunger上去,最后把所有气体推出针筒,让针筒满满都是液体。如果这时发觉不够液体,可以再提取,由于之前以把气体弄出,再提取就不会再有泡泡了。

在注射之前,这些全部要准备好。针头的盖子也要先弄松掉,不然一会儿单手是很难做这些的。然后就开始restraint老鼠,记得要看清楚身份证才抓,不要抓错老鼠打错针,哈哈。

老鼠抓好后,让鼠头往下,这样子腹腔里的内脏会比较靠向头部,就可以防止把液体打入腹腔里的内脏了。我一般习惯打老鼠右边的腹腔,因为我用右手打针,另外也是因为胃和肠会倾向于左边。打针的部位是lower right quadrant。其实老鼠身体很小,所谓lower right,也就那几个位置而已。如果你打的是母老鼠,那些地方有乳头,就是差不多对了。先用一点酒精擦擦要打的部位。

大概把针头倾斜一个45度(相对于老鼠的身体),不要打太靠近midline,因为那里有膀胱等。插进去的时候最好把针头尖的地方向下(所谓的bevel up),这样就比较尖利,比较容易进去。

http://research.uiowa.edu/animal/mp16.jpg

http://ihome.ust.hk/~webacf/files/Mice/mice-tech.ppt

最好是能把拿针筒的手指靠在另外一只手上面,有一个支撑,就不用凌空。凌空容易手抖。插进去不用很深,然后开始往后拉plunger。往后拉的目的是要确保不要刺到肠子或者膀胱,因为如果刺进去这些地方,往后拉会看到液体或半固体出现在针筒里,这时就知道打错地方了。真正在腹腔里面的话,往后拉不会拉出什么东西来的,会出现有点像真空的拉力在针筒里面。

如果打错地方了就要把针拔出来再打一次。如果确定了在腹腔里面的话,就可以开始注射(推plunger)了。注射的时候速度不快不慢,最重要是不要紧张,不要手抖,不然你要推plunger变成推整支针,针就会越插越近,伤害到老鼠。

如果针插得不够进,就会变成皮下注射(SC)了,这时你会看见你所注射的东西慢慢在老鼠的皮下累积成一个小包,这就代表打错了,可以把针拉出来再试。

打完之后放开老鼠的时候要谨慎一点,记得先抓住尾巴才松开它的颈项。

一般这样打针如果熟练的话,不用5分钟就可以了,而且一般不需要别人帮忙,可以独自完成。但如果不熟悉,可能需要不少训练。所谓熟能生巧,真是千古不变的道理啊。




Venus

注:

http://www.pubmedcentral.nih.gov/pagerender.fcgi?artid=377659&pageindex=1 - 这里有一个研究,看IP打针错误的频率和一般打错的位置。

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=5418953 - 这也是一篇说IP打针位置的文章。

http://www.rit.edu/~gtfsbi/hytc/injection.htm - 解释IP的步骤。

http://www.ibms.sinica.edu.tw/html/sup2/tech_sup.html (中文)- 各种注射方法的概括

http://research.uiowa.edu/animal/?get=mse_tech - 各种注射方法的概括

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