Background: Anxiety is a generic term for fear, restlessness, stress and can be induced by systemic disturbances or external stimulus including Nicotine and Caffeine, the widely consumed psychostimulants. In rats, anxiety can be tracked using non-invasive modalities like behavior, ECG, HRV and corticosterone. Application of non-invasive diagnosis seems pertinent in laboratory animals to ensure procedural analogy with human and balance human utilitarianism with laboratory animal welfare subsequently fostering one health concept. 
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Methods: Three groups (n=6) of adult male Wistar rats were made. Group I: control injected with saline. Group II: 0.5 mg/kg body weight (BW) nicotine and 10 mg/kg BW caffeine. Group III: 0.25 mg/kg BW nicotine and 5.0 mg/kg BW caffeine. For assessment of anxiety, recording was done immediately and on the third day of drug administration. Cognition recorded on elevated plus maze and Y maze; a specially designed non-invasive clip electrode used for ECG recording and HRV evaluation. Novel method proposed using blue tetrazolium dye for fecal corticosterone assay. 
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Result: The immediate results demonstrate increased activity and arousal behavior. However, on the third day anxiety was induced. The ECG data showed increased heart rate and QTc interval on both occasions, but sympathovagal attributes SD1/SD2 of HRV were significantly reduced on third day. The tetrazolium assay showed a significant increase in fecal corticosterone concentration on the third day. These results show that nicotine and caffeine in combination induce anxiety and can be assessed using non-invasive modalities analogous to methodologies used for human clinical diagnosis.

Non-invasive Assessment of Anxiety using Cognition, ECG, HRV, Fecal Corticosterone Biomarker in Rats exposed to Combined Acute Dose of Nicotine and Caffeine