2.7. Cognitive behavioral test: novel object recognition test (NOR)

The novel object recognition test (NOR), was used to assess the cognitive status (recognition memory) of the experimental mice and was performed as previously described [47]. This test is divided into 3 consecutive days (Fig. 3G); on the first day (T0), the experimental mice are placed in a 40 cm × 40 cm x 40 cm cage for 10 min. The next day (T1), the mice are placed in the same cage but with two identical objects in shape and color. The test itself (T2) is performed 48 h after the habituation test. In order to asses if the animals have cognitive decline, one of the objects is changed for a different one, both in shape and color. The amount of time spent exploring the novel (TN) or familiar (TF) object was recorded and the differences were represented as Discrimination Index (DI). Mice have an innate preference for novelty, thus mice without cognitive impairment will recognize the familiar object and will spend most of the time at the novel object. DI allows discrimination between the novel and familiar objects and it is calculated as follows DI = (TN-TF)/(TN + TF). For this test, the LPS administration was given just after T1 (second day).

Aged WT mice treated with LPS showed increased oxidative stress, ferroptosis and cognitive decline, but these alterations were prevented in HMOX1^M-KO mice. (A) Representative images in whole hippocampal slices of reactive oxygen species (ROS) measured with the fluorescent probes H₂DCFDA, dihydroethidium (DHE) and hippocampal cell death, measured with the fluorescent dye propidium iodide (IP). (B) Quantification of H₂DCFDA mean intensity, (C) DHE mean intensity and (D) propidium iodide mean intensity in whole hippocampal slices. Representative images (E) and quantification (F) of GPX4 (red), a ferroptotic marker. (G) Schematic representation of the novel objet recognition test (NOR). LPS (0.5 mg/kg) was injected just before day 2 (T1) and 24 h after, the test (T2) was performed. (H) Discrimination index (DI) in control and LPS treated mice. WT mice treated with LPS showed an increase in cognitive decline, measured as a reduction in the DI, which was restored in HMOX1^M-KO mice treated with LPS. Data represent mean ± S.E.M. (N = 3–12, one-way ANOVA followed by Tukey post-hoc test). Significant differences were considered when: *p < 0.05 and **p < 0.01 compared to aged WT saline mice or ##p < 0.01 compared to aged WT LPS injected mice. Scale bar = 1000 μm (A) and 10 μm (E). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)