Materials and Reagents

1. Animals
   C57bl/6J mice (Charles River Laboratories) at PND4-5
   The day of birth is defined as PND1
   The body weight of PND 5 pups should be 2.0 ± 0.2 g
   
2. Anesthetics
   
   1. Isoflurane (Vetflurane, catalog number: 137317 )
      
   2. Xylocain (local anesthetic) (AstraZeneca, catalog number: 153270 )

Equipment

1. Suture
   6-0 silk suture attached to a needle for skin closure (Vömel, catalog number: 14719 )
2. 7-0 silk suture cut into 1.0 cm–1.5 cm pieces for artery ligation (Ethicon, Johnson & Johnson, catalog number: EH7464G )
3. Needle for skin suture
4. Micropore surgical tape (Sollentuna, catalog number: 1530-0, 3 M )
5. Syringe, 1 ml (B. Braun, Omnifix® 100 Solo)
   
6. Heating plate (Labotect, model: Hot plate 062 )
   
7. Forceps (Figure 1):
   Fine-tipped curved forceps for dissecting the artery (Dumont, tweezer style 7, catalog number: 0302-7-CO )
   Fine-tipped straight forceps for dissecting the artery (Nopa instruments, catalog number: AB 501/05 )
   Hooked forceps for lifting the artery (Dimeda Instrumente GmbH, catalog number: 42.883.07 )
   
8. Fine-tipped scissors (sharp) (Dimeda Instrumente GmbH, catalog number: 08.340.09 )
9. Nasal mask for administering anesthetic gas (Simtec Engineering, catalog number: 10025 )
   
10. Anesthetic vaporizer (Ohmeda Isotec Isoflurane Vaporizer)
11. Surgical microscope (Carl Zeiss, model: S100/OPMI pico )
    
12. Water bath with adjustable temperature (Grant, model: W14 )
    
13. Hypoxic chamber with controlled temperature, humidified airflow, and variable oxygen level (Figure 2)
    Note: In this protocol, a Dräger transport incubator 5400 and a Gant type W14 water bath (Figure 2A) were used to keep the ambient temperature at 36 °C. A smaller customized incubator, the hypoxia chamber, was kept inside the transport incubator (Figure 2B). Pups were placed inside the hypoxia chamber during the hypoxia procedure. The air and the 10% oxygen were introduced into the bottom of the customized incubator and vented from the top of the incubator. Before being introduced in the hypoxia chamber, the air and the 10% oxygen mixture were humidified and heated to 36 °C with the heated water bath. The customized hypoxia chamber was 19 cm in diameter and 19 cm in length (Figure 2C).
    
    
    Figure 1. Surgical tools. Hooked fine-tipped forceps for lifting up the artery (A), fine-tipped straight forceps (B) and curved forceps (C) for dissecting the artery, and fine-tipped scissors for the skin incision (D).
    
    
    
    Figure 2. Equipment used for hypoxia treatment. A. Water bath and transport incubator containing the hypoxia camber. B. The hypoxia chamber inside the transport incubator, and (C) the dimensions of the customized hypoxia chamber.
    
    
    
    Figure 3. Hypoxia-Ischemia procedure time line
    

Procedure