The Behavioral and Neuroinflammatory Impact of Ketamine in a Murine Model of Depression and Liver Damage

Abstract

Non-alcoholic fatty liver disease (NAFLD) has been associated with depression and inadequate response to antidepressants. While ketamine has demonstrated efficacy in treating depression, its impact on pre-existing liver injury and depression remains unclear. This study aimed to evaluate the effects of ketamine treatment in a murine model of depression and liver damage, considering age-related differences. Young and aged male C57BL/6N mice were submitted to chronic unpredictable mild stress (CUMS) and methionine-choline-deficient (MCD) diet to induce depressive-like behavior and NAFLD. Behavioral testing (sucrose preference test, open field test, novel object recognition test, Crawley's sociability test) were used to assess ketamine's (50 mg/kg) effect on behavior. Hepatic ultrasonography was utilized to evaluate liver status. The cortical and hippocampal NeuN+, GFAP+, and Iba1+ signals were quantified for each animal. Ketamine administration proved effective in relieving anhedonia and anxiety-like behavior, regardless of liver damage. Although ketamine treatment did not improve memory in animals with liver damage, it enhanced sociability, particularly in aged subjects. The acute administration of ketamine did not affect the severity of liver injury, but seems to affect astrogliosis and neuronal loss. Although animal models of depression only replicate certain clinical features of the condition, they remain valuable for evaluating the complex and varied effects of ketamine. By applying such models, we could demonstrate ketamine's therapeutic versatility, and also indicate that responses to the treatment may differ across different age groups.

Keywords: CUMS; MCD diet; NAFLD; behavior; depression; ketamine.

Figure 1

The weekly experimental design of the present study included a 3-day acclimatization period, followed by one week of behavioral testing and abdominal ultrasonography, and then one week of normal habituation before the initiation of the CUMS protocol and MCD diet. In week 5, the behavioral and imaging assessments were repeated, and the ketamine treatment was administered at the beginning of week 6. All measurements were repeated two weeks later, in week 8. (A). Body weights and ultrasonography severity scores (B–E). The graphs show mean values ± SD, # p < 0.05, ## p < 0.01, and ### p < 0.001 displaying differences between sessions.

Figure 2

Behavioral assessments. Ketamine treatment led to an increased sucrose preference for (A) all young ketamine-treated animals, regardless of liver injury. After ketamine administration, CUMS + K, CUMS + MCD, and CUMS + MCD + K animals exhibited increased sucrose preference compared to CUMS mice. In (B) aged animals, SPT revealed increased sucrose preference for ketamine-treated mice, regardless of the diet, with the CUMS + MCD + K group presenting the highest preference index (88.90 ± 2.52%). OFT performed in (C) young animals showed that both ketamine and normal diets led to decreased anxiety-like behavior. Conversely, in (D) aged mice, the CUMS + MCD group displayed decreased anxiety-like behavior compared to CUMS + K and CUMS animals. The NORT of (E) young CUMS + MCD and CUMS + MCD + K mice revealed decreased preference for novel object compared to CUMS animals. No differences were observed in (F) aged animals. Crawley’s sociability test revealed decreased preference for social novelty for (G) young CUMS mice compared to CUMS + MCD and CUMS + MCD + K. In (H) aged animals, sociability increased after ketamine treatment, regardless of diet, with CUMS + K displaying the highest preference index (73.46 ± 4.45%). The graphs show mean values ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 displaying differences between treatments, and # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 displaying differences between sessions.

Figure 3

Age-related differences in behavioral tests. (A) The SPT performed after treatment and normal food revealed increased sucrose preference in young CUMS + K mice compared to aged ones. Young CUMS + K mice also exhibited reduced anxiety-like behavior in (B) OFT when compared to aged counterparts. Regardless of treatment administration, all young MCD-fed mice displayed a decreased preference for novel objects in (C) NORT at the end of the experiment. In a (D) social novelty test, aged mice fed a normal diet and submitted to the CUMS procedure showed increased preference for social novelty compared to young groups, and MCD food led to decreased index in all aged, compared to young, animals, regardless of treatment. The graphs show mean values ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 4

Immunohistochemical detection of NeuN-positive neurons in (A) the cortex revealed age-related differences, and increased NeuN+ signal area for young CUMS + MCD + K animals, compared to CUMS + K and CUMS + MCD mice. Similar age-related differences were observed in the (B) hippocampus, displaying a reduction in the cortical NeuN+ signal in all aged subjects compared to their younger counterparts. Age-related differences were also observed when analyzing both (C) cortical and (D) hippocampal area of GFAP-positive astrocytes, with increased signal observed in aged animals. (E) Neurons, astrocytes, and cell nuclei labeled with NeuN (green), GFAP (red), and DAPI (blue) for aged and young animals. The graphs show mean values ± SD, * p < 0.05, ** p < 0.01. Scale bar 50 µm.

Figure 5

Immunohistochemical detection of Iba-1-positive microglia in (A) the cortex showed no differences in young animals, but decreased Iba1+ signal for all aged animals fed an MCD diet, regardless of ketamine treatment. No significant effect on the area of Iba1+ signal was observed in (B) the hippocampus. (C) Microglia and cell nuclei labeled with Iba1 (green) and DAPI (blue). The graphs show mean values ± SD, * p < 0.05, ** p < 0.01. Scale bar 50 µm.