Most basic and translational work comes from anesthetized animals. Anesthetics can add confounding variables to data analysis and interpretation. This work enables awake imaging in rats to circumvent that issue.
This design is quite simple. The whole assembly is lightweight, so it does not impair the normal life of the animal. This means it can also be used for long-term chronic imaging.
Helping to demonstrate this procedure will be Dr.Mehwish Bhatti, a senior postdoc from our laboratory. Begin by making the head implant using CAD software to image the area posterior to the bregma and adjacent to the midline centered on the somatosensory cortex. Ensure that the head implant covers an area of 0.9 to 1.9 millimeters on the skull away from the imaging area.
Use only three screws to anchor the head implant on the rat skull. Design all the screw holes on the opposite side of the midline in the contralateral part of the image hemisphere. To allow wires to fix the head cap to the head implant, place a hollow bar in the upper part of the head implant.
Next, design the cap, ensuring that it covers the imaging area entirely and protects it from any trauma. Add a curvature to the head cap so that it aligns to the shape of the head without causing difficulty to the animal's daily activities in the standard enriched cages. Cut the inner side of the head cap in a wider rectangular shape so that the upper part of the head implant can fit into it.
Perpendicular to this rectangle, cut two other rectangular regions to anchor the head cap to the head implant. To fix the head cap on the rat's head, pass the wires through the upper hollowed bar of the head implant. Design the head frame so that one cut part can move through the upper bar of the head implant and is fixed using a clamp.
To make the contralateral side accessible for imaging, angle the other cut part to provide extra strength for keeping the rat head fixed. Print the head implant and head cap using the 3D printer. Measure the weight of the animal to calculate the amount of anesthetic to be administered.
Anesthetize the animal by injecting sodium pentobarbital intraperitoneally. Several anesthesia alternatives can be used in this surgery such as isoflurane, ketamine, or xylazine. Inject atropine intramuscularly to reduce mucus secretions while breathing.
Using a hair trimmer, shave the head of the rat starting between the eyes to the back of the ears five millimeters centered around the midline. Then monitor the partial oxygen saturation and heart rate using a pulse oximeter and heart rate monitor probe secured to the rat's hind leg. Wipe the rat's head and the surrounding area thrice with Betadine and 70%alcohol wipes alternatively.
Fix the rat in a stereotaxic system. Next, insert a petroleum jelly lubricated rectal probe to measure the rat's body temperature and maintain it through the heating blanket's feedback system to avoid hypothermia after anesthetic administration. Apply ophthalmic ointment to both eyes to prevent drying.
Administer 2%local anesthetic subcutaneously over the surgical site and three millimeters of lactated Ringer's solution at room temperature subcutaneously to prevent dehydration and provide nourishment during surgery. Using sharp surgical scissors, remove the part of the skin over the surgical site. Dissect and remove part of the skin between the ear and eye on the temporal part of the head.
Using a scalpel, remove the underlying skin tissue to expose the skull. Then clean the skull using sterilized cotton gauze. Cut the temporal muscle and expose the skull area on the contralateral hemisphere for the head implant.
Place the head implant on the skull to ascertain the location of anchoring screws for the implant. Now mark the skull using India ink for drilling the screws with the drill bit one. Drill the burr holes for the screws using dental drill bit three and screw the head implant in place.
Dry the skull using sterile gauze and apply a thin layer of tissue adhesive around and beneath the head implant to glue it to the skull. Use dental cement to support the head implant in place further and let the cement dry for two to three minutes. Further, use dental drill bit three to decrease the thickness of a 7.5 millimeter by 7.5 millimeter area on the left side of the skull just posterior the bregma and lateral to the midline to 50 micrometers.
Apply topical antibiotic ointment over the surgical site and then cover it with a thin layer of silicone rubber to protect the thin skull. Next, cover the surgical site using the head cap and fix it in place with the two small pieces of wires going through both the head implant and the head cap. Apply silicone rubber to cover the head cap and skull to stabilize the head cap further on the rat's head.
Administer buprenorphine and antibiotics intraperitoneally to manage pain and inflammation and prevent in infection respectively. The optical imaging signals from the summed response of an awake rat and a single trial of an anesthetized rat are shown. The functional response of the awake head fixed rat is 140 times stronger than that of the anesthetized rat.
Be careful not to add too much cement over the head plate. It can obstruct the cap from being properly fixed. This method can be widely applied to awake head fixed thin skull neuroimaging.
With craniotomies, it can be extended to other neuroimaging protocols including functional ultrasound imaging, calcium imaging, and fluorescence imaging. Awake imaging will allow for a new body of work without the confounding variables introduced with anesthetics.
