Abstract: BACKGROUND: Stable animal insomnia models serve as crucial tools for investigating insomnia mechanisms and developing anti-insomnia drugs. The combination of chemical and physical methods provides an effective approach for establishing animal insomnia models. 
OBJECTIVE: To compare chemical methods (p-chlorophenylalanine), physical methods (water environment sleep deprivation method), and combined methods (p-chlorophenylalanine combined with water environment sleep deprivation method) for constructing insomnia mouse models, thereby exploring the optimal modeling method.
METHODS: C57 mice were randomly divided by sex into the following groups: control (untreated), p-chlorophenylalanine, water platform, and p-chlorophenylalanine+water platform, with 12 mice per group (equal numbers of males and females). Mice in the p-chlorophenylalanine group received intraperitoneal injections of 400 mg/kg p-chlorophenylalanine suspension daily for 3 consecutive days; those in the water platform group were placed in a water environment sleep deprivation platform for 3 consecutive days; and those in the p-chlorophenylalanine+water platform group were placed in the sleep deprivation platform and received intraperitoneal injections of 400 mg/kg p-chlorophenylalanine suspension for 3 consecutive days. After 3 days of modeling, mice were subjected to the open field test, and the number of neurons in the hypothalamus and hippocampus was examined via Nissl staining. Serum levels of 5-hydroxytryptamine, gamma-aminobutyric acid, and dopamine were measured using enzyme-linked immunosorbent assay.
RESULTS AND CONCLUSION: (1) Compared with the control group, male mice in the three model groups exhibited an upward trend in total distance, number of entries into the central area, average speed, and number of stand-up episodes. The most pronounced changes were observed in the p-chlorophenylalanine+water platform group (P < 0.05 or P < 0.01). Female mice in the three model groups also showed an upward trend in total distance, number of entries into the central area, average speed, and number of stand-up episodes. (2) All three modeling methods could reduce the number of neurons in the mouse hypothalamus and hippocampus, with the highest reduction and most severe neuronal damage observed in female mice in the p-chlorophenylalanine+water platform group. (3) Compared with female mice in the control group, changes in three serum indicators were more pronounced in the female mice in the p-chlorophenylalanine+water platform group. 5-Hydroxytryptamine and gamma-aminobutyric acid levels were significantly reduced 
(P < 0.05), while dopamine levels were significantly elevated (P < 0.01). To conclude, the insomnia model established in female mice using p-chlorophenylalanine combined with the water platform is relatively more reliable and stable, providing an ideal animal model for insomnia research.

Key words: insomnia, animal model, p-chlorophenylalanine, water environment sleep deprivation, dopamine, mouse