Most project license forms require a prospective assessment of the severity of the procedure (Article 15, Directive 2010/63/EU). Stereotaxic surgeries in rodents are typically procedures that may cause more than momentary or slight pain. As such, they should only be performed with appropriate sedation, anesthesia and analgesia. In general, surgical or other painful procedures are not permitted on unanesthetized animals paralyzed by chemical agents. Whether the surgery is included in a survival procedure or is a terminal one, the rank of pain/severity will be the same. In the USA, both survival and non-survival procedures refer to the same USDA¹¹ category D: animals used for teaching, research, experiments, testing, or surgery that will involve accompanying pain or distress and for which appropriate anesthetic, analgesic, or tranquilizing drugs will be used.¹² In Europe, procedures are now classified as “non-recovery,” “mild,” “moderate,” or “severe.” The assignment of a severity degree shall be based on the most severe effects likely to be experienced by an individual animal after applying all appropriate refinement techniques. A procedure that includes a single stereotaxic surgery with a craniotomy should be ranked “moderate” (surgery under general anesthesia and appropriate analgesia, associated with postsurgical pain, suffering, or impairment of general condition (Section III of Annex VIII, Directive 2010/63/EU)). If the surgery is expected to result in severe or in moderate but persistent postoperative pain, suffering, or distress or is likely to cause severe impairment of the well-being of the animal, the procedure shall be classified as “severe” (equivalent USDA Category E¹³). For example, this can be the case if anesthetic, analgesic, or tranquilizing agents adversely affect the procedure and its results or interpretation and, therefore, cannot be used. For such procedures, the withholding of analgesia, anesthesia, or other treatment to alleviate pain/distress will have to be scientifically justified: in particular, the experimenter must provide the methods or means used to determine that pain and/or distress relief would interfere with the results. Note that for such severe/category E procedures, federal/state/local laws or policies may require separate declarations/forms and a systematic retrospective report.

Besides surgical pain, stereotaxic surgeries like other invasive techniques can induce transitory or even permanent functional damage or handicap. Depending upon the target area, or the way chosen to reach it, functional damage may be created by the sole introduction of a foreign object into the brain. To some extent, such damage can often be anticipated. For instance, if the procedure aims to lesion a brain area involved in motor control, a functional consequence should be expected that would affect locomotion or movement coordination and therefore compromise the animal’s well-being. For instance, neurotoxin lesioning of dopaminergic neurons of the substantia nigra by 6-hydroxydopamine (6-OHDA) in rats (Schwarting and Huston 1996a, b; Kirik et al. 1998) induces a postural asymmetry and a unilateral akinesia when performed unilaterally. Partial or complete lesions are often accompanied by a spontaneous or induced sensitization of locomotor activity that can acutely or chronically affect the animal’s ability to eat, drink, and behave normally (Ungerstedt 1971). In such cases, adequate postoperative care procedures such as manual feeding can be required until the animal recovers its autonomy and a complete restoration of its behavioral repertoire. In a new project, a preliminary study with few animals can be included to tentatively determine if the procedure can cause a potential handicap.

In general, only one major survival surgery (i.e., having the potential for producing a permanent handicap for an animal expected to recover from surgery) may be performed per animal. If multiple survival procedures are essential parts of the project, experimenters are expected to provide a thorough justification and to describe the chronology of the multiple surgeries, how they are interrelated, and why they are necessary to achieve the scientific objective. Here again, cumulative pain, distress, or functional deficit that may result from the succession of procedures should also be detailed.

When assessing a level of pain and distress prior to an experiment, the entire lifetime experience of the animal should be considered, not just the animal’s experience during the stereotaxic surgery. It is therefore crucial to draw attention to the other factors that can also affect the animal’s well-being, such as transportation, housing, feeding and handling, and individual history if the animal has already been used in previous experiments. Procedures or treatments that precede the surgery (e.g., operant conditioning, irradiation, food/drink restriction, environmental distress, forced exercise, disease conditions, exposure to predator scents, and excessive noise) are other factors to consider. One needs to identify as many potential sources of pain and distress as possible, their intensity, and duration. In addition to the gestures performed during the surgery itself, and among other sources, incorrect tissue manipulation, inexperience of the experimenter, inadequate postoperative care, or insufficient acclimatization of the animals are potential sources of pain or distress. Furthermore, the phenotype, particularly in genetically modified animals (transgenic, knockout, knockin, floxed, cloned, or otherwise genetically modified strain) should not be neglected. For instance, some rat or mouse strains can be prone to seizures, and additional complications may arise from this condition. Listing all potential sources of pain or distress in the experimental model will not only help in the prospective assessment of pain/distress but also serve to define additional gestures and care of the subjects that will be proposed to the committee and performed by the collaborators of the study and the animal caretaker.

After assessing the level of pain and distress in their models, experimenters are asked to thoroughly describe the methods and means used to monitor and to alleviate them.