This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.
Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.
The pig has been well documented and accepted as a translatable animal model for research in cardiovascular systems1, gastrointestinal systems2, nutrition3,4, diabetes5, toxicology6, and surgical techniques7. Use of the pig in neuroscience is beginning to increase, as PubMed searches for the keywords "swine brain animal model" result in four-times more results from 1996-2005 than the preceding 10 year period8, and even more results at present. A primary reason that the popularity of the pig model is expanding is due to its similarities in growth, structure, and function of the brain when compared with humans. In comparison to the human brain, the pig brain exhibits similar gyral patterning, vascularization and distribution of gray and white matter9. Moreover, the pig brain has been used in neuroimaging procedures, evoked potential recording, and in establishing neurosurgery techniques8,9. Unlike other animal models, however, the pig and human experience perinatal brain growth spurts, as opposed to pre- or post-natal growth spurts. At birth, the human and pig brain weigh approximately 27 and 25 percent of their adult brain weight, respectively, compared to the rat brain that weighs 12 percent of its adult brain weight and the rhesus monkey brain at 76 percent of adult weight10.
One reason the pig has been only slowly adopted as an animal model for neuroscience is because many researchers are unfamiliar with the animal in this context. Researchers may not be aware of its potential uses in the field or may not know the proper techniques required to use such a model. As use of the pig as a biomedical and preclinical model gains attention and use in neuroscience, it is necessary to establish standardized procedures of tissue removal to ensure accurate comparison of data across studies. Although dissection and surgical techniques involving the pig brain have been published elsewhere11,12,13, there is a need for simple and standardized protocols to collect pig brain tissue, especially for use in biochemical assays. As such, the aim of this video is to provide the knowledge necessary to allow researchers to perform a standardized brain extraction and dissection. This video illustrates one proper technique to remove the pig brain while keeping the cortex and brainstem intact, and subsequently review methods to dissect several key brain regions.
Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois at Urbana-Champaign
NOTE: Prior to euthanasia, the pig was anesthetized via intramuscular injection with a combination of telazol:ketamine:xylazine (50.0 mg of tiletamine HCl plus 50.0 mg of zolazepam HCl reconstituted with 2.50 mL of ketamine HCl (100 g/L) and 2.50 mL of xylazine (100 g/L) and administered at 0.06 mg/kg BW). Once anesthetized, the pig was euthanized via intracardiac administration of sodium pentobarbital (390 mg/mL administered at 1 mL/5 kg BW). For brain dissection, it is recommended that the method of euthanasia be chosen based on the desired analytical procedure of the tissue. The method of euthanasia should cause as little damage to the brain as possible.
1. Extraction of the pig brain
2. Dissection of the pig brain
NOTE: It may be helpful to use a brain atlas or fiber dissection guide14 as a visual representation during dissection procedures. Make sure dissected tissue samples are stored properly according to project-specific needs upon removal of each sample (described in more detail below). Additionally, please note that for the purposes of this video, all brain regions shown were dissected from the right hemisphere, but this may differ per laboratory based on experimental objectives.
3. Post-dissection
This section describes examples of results obtained after correct extraction and dissection of a 4-week-old pig brain. Figure 1 outlines the shape of each brain region for use as a guide during dissection. Part of the brainstem may remain in the skull after removal of the cerebellum (Figure 1B). This can be removed while isolating the desired region of the cerebellum. Table 1 displays the average weight (mean ± standard error of the mean) for each of the dissected brain regions (n=5).
Figure 1: Extracted Pig Brain. Outlines of brain regions for use as a guide during dissection. Regions shown are from the right hemisphere. Please click here to view a larger version of this figure.
Region | Weight (g) | SEM |
Whole Pig* | 8.006 | 0.545 |
Brainstem | 0.829 | 0.132 |
Cerebellum | 5.929 | 0.137 |
Midbrain | 0.376 | 0.047 |
Hippocampus | 0.500 | 0.051 |
Striatum | 0.410 | 0.115 |
Thalamus | 0.476 | 0.120 |
medial Prefrontal Cortex | 0.459 | 0.122 |
*Weight presented as kg |
Table 1: Brain Regions Weights. Average weight of the 4-week-old pig brain and each dissected brain region (n=5).
The techniques described herein were designed for pigs approximately 4 weeks of age. It is critical to perform these steps immediately after the pig has been humanely euthanized to ensure the integrity of brain tissue structure is maintained, especially when considering subsequent biochemical assays. It is helpful to use an atlas or fiber16 dissection guides when first learning the techniques. It is recommended that the experimenter practice several brain extractions and dissections prior to obtaining samples for data collection. The most difficult step is removal of the skull. This step will become easier with experience as it largely requires firsthand practice to know where on the skull to saw and when the skull has been cut through. This procedure is similar to that described by Bassi et al.12, though it does not require the need to create a hexagonal cranial window and provides a visual tutorial of how to perform the technique.
A limitation of this technique it that when working with older pigs, it may take more time to remove the skull as it becomes significantly thicker with age. If using a saw is too laborious or ineffective for thicker skulls, it may be necessary to either use a hammer and chisel, like that shown by Bjarkam et al.13, or powered surgical equipment (e.g., bone saw). Furthermore, this technique does not always ensure the capture of the olfactory bulbs.
The authors have nothing to disclose.
The authors would like to acknowledge Jim Knoblauch and Martin-Booth Hodges of the College of Agricultural, Consumer and Environmental Sciences Information Technology and Communication Services for their expertise in shooting, recording, and editing audio and video.
#22 Scalpel Blades for #4 Handles | Ted Pella, inc. | 549-4S-22 | |
11 1/2" Satterlee Bone Saw | Leica Biosystems | 38DI13425 | |
5 1/2" Skull Breaker with Chisel End (Meat Hook) | Leica Biosystems | 38DI37636 | |
5-inch Heavy Duty Workshop Bench Vise | Pony | 29050 | |
Butcher Knife 25cm | Victorinox | 5.7403.25 | Sharpen before use |
CM40 Light Duty Drop Forged C Clamps | Bessey | 00655BC3120 | |
Diamond Hone Knife Shaper | Chef’s Choice | 436-3 | |
Shandon Stainless-Steel Scalpel Blade Handle #4 | ThermoScientific | 5334 | |
Tissue Forceps | Henry Schein | 101-5132 | |
Vinyl Dissecting pad | Carolina | 629006 |