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Self-inflicted injury (SII) is prevalent and debilitating among adolescents. One of the most salient developmental contexts is the parent-child relationship, given its importance for shaping self-regulatory processes known to affect SII risk. A key strategy for promoting healthy family interactions is validation, or communicating an understanding of how another came to act, think, or feel. Validation appears to foster adaptive youth self-regulation, whereas chronic familial invalidation and conflict escalation are associated with emotion and physiological dysregulation and SII risk. The current study examined how teaching a validation-oriented skill from Dialectical Behavior Therapy affects behavioral and biological indices of SII risk among self-injuring adolescents and their mothers (n = 30 dyads), and typical control mother-daughter dyads (n = 30). Behavioral indicators of family functioning (e.g., cohesion, coercion, invalidation) and physiological indices of emotion regulation (respiratory sinus arrhythmia [RSA]) were examined across conflict and validation-oriented discussion tasks. Dyads’ subjective affect and observed behavior generally improved when practicing validation. Participant RSA did not significantly differ across study tasks. However, findings indicate mother-, daughter-, and dyad-level behavior accounted for significant variance in RSA reactivity. Results indicate teaching a single skill on one occasion can have detectable effects on areas of functioning relevant to SII.