A Surgical Protocol for Precise High-Throughput and Precise Viral Injections in Rhesus Monkey Brain

Clogged injection circuit: On rare occasions, the injection circuits may become clogged, either due to small fragments in the viral solution or mineral oil, or because tissue is pushed into the needle during brain penetration when targeting multiple sites within the same structure.

Alternating flushes of sterile water and 70% ethanol can often clear a clogged circuit, especially when using a pump to maintain steady positive pressure and injection flow. If clogging occurs during surgery following a tissue penetration, it is likely caused by tissue obstructing the needle tip. In this case, use a pair of fine forceps to gently wipe the tip of the needles and remove debris. Afterwards, verify that the injection flow is restored.

Seroma fluid accumulation: Accumulation of seroma is relatively common after such extensive surgeries involving dissection of multiple tissue layers, detachment of the temporalis muscles, and large craniotomies. This is due to a large area of dead space, and is more commonly seen in larger animals (e.g. males). Excessive seroma buildup typically becomes apparent in the second postoperative week.

Pay close attention to the wound closure. Ensure that the bone flap is properly repositioned so its margins are well aligned with the surrounding skull, and securely tighten the suture lines connecting the flap to the bone. To minimize dead space, suture the posterior portions of the temporalis muscles together when anatomically possible (note that there are variations and some animals, particularly females, may have less medial overlap). Close the galea as tightly as possible to prevent fluid accumulation beneath it. Maintain strict sterility throughout the surgery, keep the tissues moist, and irrigate generously before closing each layer. In cases of excessive seroma formation, consider draining the fluid, since pressure from the expanding seroma can facilitate dehiscence of the incision line and impede healing, even if the sutures remain intact. Additionally, a diuretic, such as furosemide (1 mg/kg) once daily, can be initiated to decrease fluid accumulation in consultation with a veterinarian. It is recommended to culture the fluid drained from the surgical site to evaluate for any evidence of developing infection.

Meningeal adhesion: During the healing process, the dura and pia mater can fuse together. Infection can promote adhesion formation. Moreover, mechanical trauma from needle penetrations may lead to localized meningeal adhesions. This issue is particularly important for dorsal cortical targets, as removing such adhesions during brain extraction can risk cortical damage.

To reduce the likelihood of meningeal adhesion, minimize tissue trauma during needle entry and thoroughly irrigate the surgical site to remove any blood clots before dura closure. More importantly, avoid overlapping the dura incisions with penetration sites.