Day 1

Northeast Ohio Medical University

Rootstown, Ohio, 44272

vohanyan@neomed.edu

MouseMicrosurgery workshop

We would like to thank the following companies for supporting and sponsoring this workshop

Survival surgeries

• Myocardial infarction

• Transaortic banding and de-banding

If mouse survives after the surgery, we will keep the mouse until last day of the workshop. We will do echocardiography all mice survived after surgery. Echocardiography will approve the size of the infarction and the degree of transaortic constriction.

Day 1• Day One

• 8-8:30 a.m.

• Introductions

• 8:30-9:30 a.m.

• Acute and Chronic Surgery

• Anesthetics and Analgesics

• Post-surgical care and monitoring

•

9:30-10 a.m.

• Coffee break

• 10 a.m. - Noon

• Demonstration of the following:

• Dissection of vessels in mice(femoral artery and cartoid artery)

• Mouse jugular vein catheterization

• Pressure measurement (from femoral or cartoid arteries)

• Pressure volume measurement

• Noon - 1:30 p.m.

• Lunch

• 1:30- 5 p.m.

• Practical session: Participants will select the type of vessel he or she would like to practice

• Demonstration: Femoral artery blood flow and pressure measurement; computerized data collection. Participants who are interestedcan follow this demonstration.

• 5 - 5:30 p.m.

• Questions and Answer Session

Day 1Surgical Instruments:• List of necessary instruments for mouse surgery• Care and cleaning, sterilization

Surgical Station:• Table (heated and non-heated)• Securing animals on the surgical table• Hair removal and preparation of surgical region

Microscopes:• Type of microscopes needed for the surgery• Lighting sources• Depth of field, field of vision

Acute and Chronic Surgery:• Aseptic technique, sterilization methods• Sutures and suturing techniques• Catheter material and manufacture

Anesthetics and Analgesics:• Types of anesthetic for surgery• Types of analgesics for chronic surgery• Postoperative care (chronic procedures)• Respirators and ventilators for anesthesia

Surgical tools

Care and cleaning

• Before the surgery, check the list of surgical tools you need.

• Quality is important , not quantity. Quality of surgery highly depends on quality of instruments. It saves times, animals, and money.

• During the surgery keep only the surgical tools you need. Excess of surgical tools will make your table messy and it will be hard to find the tool you need.

• Take care of your instruments. Micro-scissors dull easily. This will compromise cutting blood vessels for cannulation. Micro-forceps bend easily, and a bent forceps will not hold tissue.

• After every surgery wash and dry your instruments. Take special care during washing, otherwise it is easy to break and/or bend micro-forceps and micro-scissors.

• Use Alconox or Alcotabs. Immerse the instrument for 5-10 minutes in this solution. This will help to remove the blood from the tools.

• Use soft brush (tooth brush is good) or surgical gauze for washing.

• Keep locks and ratchets open during cleaning. After cleaning rinse your tools in deionized/distilled water.

• Store the instruments in a sterilization box, and then put them into the drying oven or autoclave using the drying cycle.

• It is best to store micro-instruments in a locked drawer as they have a tendency to develop “legs.”

Care and cleaning

Loaning your instruments to “Friends”- NEVER!!! And the answer is: -I would, if I could, but unfortunately I cannot. But if you have extra tools, always help your friends!

Care and cleaning

N E V E R !!!Curved forceps

25-$80 or more

Dumont #5 Forceps

$25-$60 or more

Spring Scissors

$270-$420 or more

A B C

D E F

Care and cleaning

Cover the tips of micro-scissors and micro-forceps with plastic or rubber caps

N E V E R !!!

AB

C D

Day 1Surgical Instruments:• List of necessary instruments for mouse surgery• Care and cleaning, sterilization

Surgical Station:• Table (heated and non-heated)• Securing animals on the surgical table• Hair removal and preparation of surgical region

Microscopes:• Type of microscopes needed for the surgery• Lighting sources• Depth of field, field of vision

Acute and Chronic Surgery:• Aseptic technique, sterilization methods• Sutures and suturing techniques• Catheter material and manufacture

Anesthetics and Analgesics:• Types of anesthetic for surgery• Types of analgesics for chronic surgery• Postoperative care (chronic procedures)• Respirators and ventilators for anesthesia

Surgical Station:

• Table (heated and non-heated)- During the surgery it is very important to monitor animals body temperature. Because of the small size, mice are losing body temperature very fast. It is very important to keep the animals in warm area after the surgery, during the recovery period. Be very carefully from overheating the animals.

• Securing animals on the surgical table - Non-obese animals you can secure on surgical table using taps(Figure 1) . Hair removal and preparation of surgical region- For hair removal ChroMini Cordless Clipper (Harvard Apparatus #341414, Figure 2A).

Chemical hair removal (Nair, from any store) can also be used. Clean the skin well after using NAIR(Figure 2B). If the NAIR stays on skin, it can cause irritation and inflammation of skin.

For survival surgery, clean the skin with 1) 70% ethanol, 2) with betadine, and 3)with 70 % ethanol again . After preparing the skin, cover the mouse with surgical towel(Figure 3).

Figure 1 Figure 2 Figure 3

A B

Day 1Surgical Instruments:• List of necessary instruments for mouse surgery• Care and cleaning, sterilization

Surgical Station:• Table (heated and non-heated)• Securing animals on the surgical table• Hair removal and preparation of surgical region

Microscopes:• Type of microscopes needed for the surgery• Lighting sources• Depth of field, field of vision

Acute and Chronic Surgery:• Aseptic technique, sterilization methods• Sutures and suturing techniques• Catheter material and manufacture•

Anesthetics and Analgesics:• Types of anesthetic for surgery• Types of analgesics for chronic surgery• Postoperative care (chronic procedures)• Respirators and ventilators for anesthesia

Microscopes:

• Type of microscopes needed for the surgery

Leica/Nikon/Zeiss/ Olympus/AmScope and many other companies have dissecting and surgical

binocular and trinocular microscopes. During microscope selection always pay attention on

working distance (more than 30 mm)

• Lighting sources(Figure 3)

• Depth of field- When working, select the largest possible working distance which will still permit

you to assume a comfortable working position. This offers the benefit of a good depth of field

and sufficient space for instrumentation

• Field of vision- is the area that is visible to you when you look through the microscope

eyepieces. When you increase the magnification, you are focusing in on a smaller area.

Figure 1 Figure 2

Fiber Optic Dual Gooseneck

Microscope Illuminator

Figure 3

Headlight+Loupes

Flip Up Loupes

Fiber Optic Microscope

Ring Illuminator

Working distance

Day 1Surgical Instruments:• List of necessary instruments for mouse surgery• Care and cleaning, sterilization

Surgical Station:• Table (heated and non-heated)• Securing animals on the surgical table• Hair removal and preparation of surgical region

Microscopes:• Type of microscopes needed for the surgery• Lighting sources• Depth of field, field of vision

Acute and Chronic Surgery:• Aseptic technique, sterilization methods• Sutures and suturing techniques• Catheter material and manufacture

Anesthetics and Analgesics:• Types of anesthetic for surgery• Types of analgesics for chronic surgery• Postoperative care (chronic procedures)• Respirators and ventilators for anesthesia

Sterilization

• Sterilization can be achieved by1. Heat (autoclave)2. Chemicals3. Irradiation4. Filtration or combination

Hot Bead SterilizersFST. #18000-45

#18000-50

Daring the surgery day you can sterilize your surgical tools using hot bead sterilizer. Before sterilizing clean your tools with 70% ethanoland remove the blood from the tools

Sutures and suturing techniques• During the surgery never use big size needles and sutures, They can cause

severe damage of tissue and skin.For closing mouse skin use-6-0 or 5-0 Ethilon®, nylon suture w/ reverse cutting (C-2) needle.-6-0or 5-0 Vicryl®, polyglactin suture w/ taper (RB-1) needle.- You can also use any tupe 5-0 or 6-0 absorbable suturesFor mouse transaortic banding use5-0 or 6-0 non-absorbable sutures with or without needels. For LAD ligation, use 8-0 Ethilon®, nylon suture w/ taper (BV130-5) needle.For femoral and carotid artery, jugular vein isolation and catheterization use5-0 or 6-0 sutures.

FST. Castroviejo Needle Holder

#12565-14#12060-01

Always use small needle holder with or without lock

#18020-60

Catheters

During catheterization always pay attention on outer and inner dimmer of the catheter. Commonly used lab catheters diameters are shown in the figure below.

Day 1Surgical Instruments:• List of necessary instruments for mouse surgery• Care and cleaning, sterilization

Surgical Station:• Table (heated and non-heated)• Securing animals on the surgical table• Hair removal and preparation of surgical region

Microscopes:• Type of microscopes needed for the surgery• Lighting sources• Depth of field, field of vision

Acute and Chronic Surgery:• Aseptic technique, sterilization methods• Sutures and suturing techniques• Catheter material and manufacture

Anesthetics and Analgesics:• Types of anesthetic for surgery• Types of analgesics for chronic surgery• Postoperative care (chronic procedures)• Respirators and ventilators for anesthesia

Mouse anestheticsInjectable anesthetics: ketamine/Xilazine (intraperitoneal injection) ; 10/1 ration is commonly used anesthetic for long term mouse surgery. If surgery takes >30 minutes, another extra dose will be administered. The other alternative to keep animals anesthetized after injection is gas(Isoflurane) anesthesia (1-2% with 100% oxygen)For short term procedures gas anesthesia can be used(isoflurane 2-3% with 100 % oxygen through nose cone).

Intraperitoneal injection

Isoflurane 3-4%= HR = 350-300

Isoflurane 1.5-2.5% HR= 550-450

kv 1.5 #138

Ch

1, B

P (m

mH

g)

-200

-100

0

100

200

hx

21:00 21:10 21:20 21:30 21:40 21:50 22:00 22:10 22:20 22:30 22:40 22:50 23:00 23:10 23:20 23:30 23:40 23:50 24:00 24:10 24:20 24:30 24:40 24:501

12:20:54.200 AM

Signs of adequate anesthesia• Adequate general anesthesia is accompanied by loss of muscle tone reflected in loss of purposeful

movements. There is loss of reflexes for example corneal, pinnae and pedal. There should be no response to aversive stimuli e.g. tail pinch, pinching abdominal skin with forceps and a lack of vocalization. There are changes in the depth and frequency of respiration and cardiovascular parameters.

Monitoring the depth of anesthesia• Assess movement, stimulus perception and reflexes - [cornea, toe, tail or ear]

• Observe chest wall movement

• Pulse, heart rate, direct or indirect blood pressure (cuff or Doppler)

• Temperature

Analgesics

• Buprenorphine (0.05-0.1 mg/kg)

Inject one dose before the surgery. Inject second and third doses of Buprenorphine (0.05-0.1 mg/kg) 12 and 24 hours after the operation (12 hours intervals).

Bupivacaine(0.2cc)

0.2cc of diluted bupivacaine [0.025%] (diluted stock 1/10 with sterile saline) around the surgical area.

After administration of this analgesics, mouse will be ready for the surgery.

Ventilators and anesthesia stations

Harvard apparatusKent Scientific

Nose cones

Video

• For more information, please visit our websites:

• http://www.neomed.edu/academics/medicine/departments/integrative-medical-sciences/microecho

• https://www.facebook.com/pages/Mouse-Microsurgery-and-Echocardiography-Workshop/398774443577599