Stereotactic Injection of Hydrogel into a Post-Stroke Mouse Model

Abstract

Source: Wilson, K.L., et al. Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke. J. Vis. Exp. (2020)

This video demonstrates the stereotaxic injection of a hydrogel into the stroke core of a post-stroke mouse, highlighting its role in scaffold formation that mimics the extracellular matrix, promotes cellular infiltration, and supports angiogenesis, underscoring its neurotherapeutic potential in stroke recovery.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

Injection of hydrogel or other biomaterials

1. Perform the injection of biomaterials at a user-defined time. In this experiment, injections were performed between 5 and 7 days post-stroke. 2 - 6 µL of hydrogel is injected.
   1. Turn on the heating pad to maintain the mouse's body temperature during anesthesia (37 °C).
   2. Prepare autoclaved scissors, forceps, cotton, vet eye ointment, surgical glue or suture material, sterile alcohol, and beta iodine wipes. Prepare the bone drill with sterile drill bit heads and the 25 µL glass Hamilton syringes with metal 30 G needles.
   3. Turn on the bead sterilization to 160 °C to allow for the sterilization of instruments between mice.
   4. Attach the injection pumps to the stereotaxic device. Set the flow rate to 1 µL/min and set the volume to 4 µL.
   5. Prepare a clean cage for the mice after surgery.
2. Place the mouse into the induction chamber with an isoflurane concentration of 4% to anesthetize it until spontaneous movement stops and its breathing comes to a slow, steady rate.
   1. Once asleep, transfer and secure the mouse to the stereotaxic device with a maintenance isoflurane concentration of 1.5%.
   2. Apply the vet eye ointment to both eyes.
   3. Shave any fur that may have regrown off the mouse’s head, and aseptically prepare the area by using an alcohol wipe followed by a beta iodine wipe. Repeat three times.
      NOTE: If necessary, use an extra alcohol wipe at the end to clean off all the beta iodine, as it causes skin irritation and leads the mice to itch their skull after surgery.
3. Using the small scissors and/or forceps, reopen the skin above the brain. Use cotton with sterile saline or PBS (Phosphate-buffered saline) to clean any debris from the skull. A white-yellow circle (the stroke, Figure 1B) and the burr hole from the drill will be visible. If the dead tissue is not visible, likely no stroke occurred. If the burr hole is not centered above the stroke, re-drill to center it.
4. Orient the injection pump over the mouse and lock it at 90°. Clean the syringe with sterile saline or PBS solution before backloading the material.
   1. Once clean, disassemble the glass syringe so that there is no needle. Backload the syringe using a 25 µL positive displacement pipette with 10 µL of hydrogel (or other biomaterial here with or without cells).
   2. Using the syringe plunger, push the gel all the way to the front of the syringe. Then, reassemble the syringe and continue pushing until the gel visibly exudes from the needle.
5. Place the syringe onto the pump. The back of the pump may need to be adjusted so the syringe fits. Adjust the flow rate to 30 µL/min, select Withdrawal or Inject depending on the direction it needs to move, and then press Start. Hit Stop when the back of the pump is at the correct location.
   1. Screw the back of the pump so the syringe is secure. If adjustments were previously made, make sure the flow rate is set back to 1 µL/min and is set to inject.
6. Move the pump in the x and y directions to orient it over the hole. Move the pump down in the z direction until the needle of the syringe touches the top of the hole.
7. Reset the Z on the digital display console. Then, move the syringe down in the z direction 0.750 mm. If the syringe bends or there is resistance, the skull either healed or was not completely drilled through, and needs to be re-drilled.
8. Press Start on the pump console to inject 4 - 6 µL of the hydrogel at a rate of 1 µL/min.
9. Set a 5-minute timer when the pump stops injecting to allow the gel to begin cross-linking.
10. After 5 minutes, slowly pull up the syringe by turning the z-nob. Watch to see if any material is accidentally pulled or leaking from the hole. Unlock the pump and move it away from the mouse when the needle is far enough away from the skull.
11. Using forceps, surgical glue, or sutures, close the skin above the head. Place the mouse in the clean cage and observe its recovery. It should take about 5 – 10 minutes for the mouse to recover from anesthesia.

Results

Figure 1: Schematic representation of bregma and localization of stroke. (A) The p...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25uL Hamilton Syringe 702RN, no needle	Fishcer Scientific	14824663	Syringes used to inject biomaterials
25uL Positive displacement pipette	Gilson	M-25
Adjustable Stage Platform	Kopf Instruments	901
C57BL/6 Mice	Jackson Laboratory	664	8-12 weeks of age
Cage Assembly Rob	Thorlabs	ER3-P4	3" Long, diameter 6mm, 4 pack - for attaching laser to sterotax
Cotton Swabs	VWR	89031-288
CP 25 pipette tips	Gilson	F148012
Heating Plate	Kopf Instruments	HP-4M
IMPAC 6-Integrated Multi Patient Anesthesia Center	VetEquip	901808
Jewlers Forceps #5	GFS chemicals	46085
Miltex Adson Dressing Forceps, Disecting-grade	Intefra Miltex	V96-118
Mini-pump variable flow	Thomas Scientific	70730-064	Pump for perfusions
Mouse Gas Anethesia Head Holder	Kopf Instruments	923-B
Nanojet Control Box	Chemyx	10050
Nanojet pump header	Chemxy	10051	Attach to stereotaxic device for injecting biomaterial
Needle RN 30G PT STY 3, 0.5 inch	Fishcer Scientific	NC9459562
Non-rupture ear bars 60º	Kopf Instruments	922
Puralube Vet Ointment	Dr. Foster Smith	9N-76855
Rectal Probe Mouse	Kopf Instruments	Ret-3-ISO
Sol-M U-100 Insuline syringe with 1/2 unit markings 0.5 mL	VWR	10002-726	To inject rose bengal
StainTray Slide Staining System	Simport Scientific	M920-2	For staining slides
Sterotaxic device	Kopf Instruments	940	Small Animal Stereotaxic Instrument
Student Adson Forceps -1x2 teeth	Fine Science Tools	91127-12
Student Fine Forceps - straight/broad Shanks	Fine Science Tools	91113-10
Temperature Controller	Kopf Instruments	TCAT-2LV
Tissue-Tek OCT compound	VWR	25608-930
Vetbond Tissue Adhessive 3mL	Santa Cruz Biotechnology	sc-361931