An Improved Surgical Approach for Complete Interhemispheric Corpus Callosotomy Combined with Extended Frontoparietal Craniotomy in Mice

Abstract

Callosotomy is an invasive method that is used to study the role of interhemispheric functional connectivity in the brain. This surgical approach is technically demanding to perform in small laboratory animals, such as rodents, due to several methodological challenges. To date, there exist two main approaches for transecting the corpus callosum (CC) in rodents: trephine hole(s) or unilateral craniotomy, which cause damage to the cerebral cortex or the injury of large vessels, and may lead to intracranial hemorrhage and animal death. This study presents an improved surgical approach for complete corpus callosotomy in mice using an interhemispheric approach combined with bilateral and extended craniotomy across the midline. This study demonstrated that bilateral and extended craniotomy provided the visual space required for hemisphere and sinus retraction, thus keeping large blood vessels and surrounding brain structures intact under the surgical microscope using standardized surgical instruments. We also emphasized the importance of good post-operative care leading to an increase in overall animal survival following experimentation. This optimized surgical approach avoids extracallosal tissue and medium- to large-sized cerebral blood vessel damage in mice, which can provide higher study reproducibility/validity among animals when revealing the role of the CC in various neurological pathologies.

Keywords: corpus callosotomy; craniotomy; interhemispheric; mice; superior sinus retraction.

Figure 1

Bone flap creation. (a) Visual explanation for (b) mice skull through the microscope. (c) Using a diamond-tipped engraver, we drilled an ellipsoid notch from the bregma point to the lambda (about 8 mm). Being careful to avoid pressure, we drilled the groove to a bone thickness of ~0.5 mm. (d) With a micro hook tip, we picked up the bone flap and carefully lifted it. For better results when separating dura mater from the bone flap, we applied a few drops of saline inside the skull groove. (e) Visualization of superior sinus and blood vessels after bone flap removal. Image (a,c) was created at BioRender.com, accessed on 5 June 2022.

Figure 2

Dura mater flap creation. (a) After removing the bone flap, there was visible dura mater and superior sinus. (b) Visual explanation for (a). (c) We carefully pinched dura mater with micro tweezers, creating flaps, and placed aside as in (d). Image (b,d) created at BioRender.com, accessed on 5 June 2022.

Figure 3

Micro hook positioning on bregma. The superior sinus was gently retracted and repositioned using a cotton swab followed by positioning of the micro hook, which had been placed, in a micro-manipulator, on the bregma between the two hemispheres within the longitudinal fissure. Top view (a,b). Side view (c,d). Image (a,c) created at BioRender.com, accessed on 5 June 2022.

Figure 4

Dorsoventral dissection. The blunt end of the micro hook was placed in the dorsoventral direction, 2.8 mm deep, inside the longitudinal fissure at the relative point nearest to the dura under the bregma. Top view (a,b). Side view (c,d). Image (a,c) created at BioRender.com, accessed on 5 June 2022.

Figure 5

Rostral-caudal dissection. The distance from the micro hook rod and bregma was measured (1.5 mm) and then moved in the rostral-caudal direction, targeting the frontal part of the corpus callosum. Top view (a,b). Side view (c,d). Image (a,c) created at BioRender.com, accessed on 5 June 2022.

Figure 6

Caudal-rostral dissection. The micro hook was placed on the bregma and moved in the caudal-rostral direction 3.5 mm from the bregma, to target the middle and rear parts of the corpus callosum. Top view (a,b). Side view (c,d). Image (b,d) created at BioRender.com, accessed on 5 June 2022.

Figure 7

Dissection schematic. A schematic overview of the cuts required for complete corpus callosotomy, including coordinates relative to bregma. A view from the top (a) and inset, magnified coronal view (b). Image created at BioRender.com, accessed on 5 June 2022.

Figure 8

Post-surgical fixation of the bone flap. Surgery was concluded by anatomically positioning the bone flap and securing it with dental cement that had been kept in a semi-liquid state via addition of solvent when required (a,b). Dental cement was applied to the bone flap/skull groove using a dental micro-curette. Dental cement should dry completely prior to wound suturing. Image (a) created at BioRender.com, accessed on 5 June 2022.

Figure 9

Co-ordinate specific Nissl-stained sections. (a) Visual representation of coordinates relative to bregma. (b) Nissl-stained sections of callosotomized mouse brain. Image (a) created at BioRender.com, accessed on 5 June 2022.