Academic Editors: Rafael Franco and Santosh Jadhav
Background: Lipopolysaccharide (LPS) administration is one of the most commonly used methods for inducing inflammation in animal models. Several animal studies have investigated the effects of acute and chronic peripheral administration of LPS on cognitive impairment. However, no previous study has compared the effects of different doses of chronically administered LPS on recognition memory performance. Aim: Here, we aimed to investigate the optimal dose of chronically administered LPS for the induction of recognition memory impairment in mice. Materials and methods: LPS at different doses (0.25, 0.50 and 0.75 mg/kg) was administered to SWR/J mice daily for 7 days. On day 9, the open field, novel object recognition and novel arm discrimination behavioral tests were performed. Additionally, prefrontal cortical histological examination was conducted. Results: Compared with the control group, mice injected with 0.75 mg/kg LPS notably showed no object preference (familiar vs. novel), a reduction in the discrimination index, and spatial recognition impairment. Administration of the 0.25 and 0.50 mg/kg doses of LPS showed a preference for the novel object compared with the familiar object, had no significant impact on the discrimination index, and caused spatial recognition impairment. These behavioral results are in line with the histological examination of the prefrontal cortex, which revealed that the 0.75 mg/kg dose produced the most histological damage. Conclusions: Our findings suggest that for chronic peripheral administration of LPS, 0.75 mg/kg is the optimal dose for inducing neuroinflammation-associated recognition memory deficits.
Neuroinflammation is defined as the immune response within the central nervous system (CNS) and is characterized by stimulation of neuroglial cells and cytokine production . Neuroinflammation occurs naturally as a neuroprotective mechanism to repair damaged neural cells . Moreover, neuroglial cells such as microglia and astrocytes play a pivotal role in neurogenesis and cell survival . However, studies suggest that prolonged brain inflammation has a neurodegenerative effect [4, 5]. There is accumulating evidence that uncontrolled neuroinflammation has a substantial pathogenetic role in various neurodegenerative disorders, including Parkinson’s disease (PD), multiple sclerosis (MS) and Alzheimer’s disease (AD), which are associated with memory deficits and behavioral alterations .
Several animal studies have demonstrated a link between neuroinflammation and recognition memory impairment [7, 8, 9]. Recognition memory can be referred to as the brain’s ability to assess the familiarity for an item in addition to the recollection of specific experienced events . Episodic recognition memory neuroimaging studies reveal retrieval-associated activation of the right prefrontal cortex (PFC) [11, 12]. Moreover, another study showed that the PFC plays a key role in both component processes of recognition memory, i.e., familiarity and recollection . Rodent behavioral and electrophysiological studies suggest that the medial prefrontal cortex (mPFC) is critically involved in recognition memory [10, 14].
Mouse models have been generally used to investigate basic diseases mechanisms and for preclinical drug evaluation . Lipopolysaccharide (LPS) administration is one of the most commonly used experimental animal models for studying behavioral and neurochemical alterations caused by neuroinflammation . LPS is a stable and potent endotoxin that resists enzymatic breakdown and induces a long-lasting inflammatory response . LPS at different doses and injection frequencies has been used to trigger cognitive impairment [16, 18, 19]. Moreover, studies have shown that intraperitoneal (i.p.) injection of LPS leads to cognitive impairment in rat and mouse models [16, 20] in various behavioral paradigms, including spatial memory, fear conditioning and recognition memory performance [21, 22, 23]. Salmani et al.  reported that male BALB/c mice treated for 7 days with 0.25 mg/kg LPS, i.p., spent less time exploring the novel object compared with the control group. Another study demonstrated that acute peripheral injection of 0.50 mg/kg LPS decreased preference for the novel object in mice . In addition, Frühauf et al.  showed that 0.75 mg/kg LPS administered for 7 consecutive days reduced preference for the new object in the novel object recognition task in Swiss mice. However, to the best of our knowledge, there is no study comparing the effects of chronic administration of different LPS doses on PFC neurodegeneration or recognition memory performance. Therefore, in this study, we aimed to determine the optimal dose of chronically administered LPS for the induction of recognition memory impairment in mice.
In this study, 24 adult male Swiss mice (SWR/J) weighing 18–25 g were obtained
from the animal facility of the King Fahd Medical Research Center (KFMRC), King
Abdulaziz University, Jeddah, Saudi Arabia. Mice were housed three per cage under
controlled temperature (23
LPS (E. coli O111: B4) was obtained from Invivogen, France. A stock
solution of 5 mg/mL LPS was prepared by dissolving 5 mg of powdered LPS in 1 mL
of endotoxin-free water, and divided into 100
Mice were randomly divided into the following four groups (six animals per group): (I) i.p. saline vehicle (0.9%) (control) group; (II) i.p. LPS (0.25 mg/kg) group; (III) i.p. LPS (0.50 mg/kg) group; and i.p. LPS (0.75 mg/kg) group. All treatments were administered for 7 consecutive days between 11 AM and 12 PM. Behavioral tasks were conducted from day 9 to 12. On day 13, the mice were sacrificed, and the brain tissues were collected for further analyses (Fig. 1A).
Timeline and design of the experiments. (A) Total duration of the study was 13 days. On the first 7 days, LPS administration was conducted daily. Behavioral tests were performed from day 9 to 12. Then, mice were sacrificed on day 13 and the brains were removed for histological examination. (B) NORT phases. The familiarization phase is designed to give the mice the chance to explore two identical familiar (F) objects. The test phase is designed to investigate the ability of the mice to recognize the novel (N) object. (C) NADT phases. The first trial is designed to allow free exploration in only 2 arms of the Y maze after blocking the novel arm (NA). The second trial evaluates the ability to recognize and explore the previously blocked arm. LPS, lipopolysaccharide; OFT, open field test; NADT, novel arm discrimination task; NORT, novel object recognition task; F, familiar object; N, novel object; SA, starting arm; NA, novel arm; OA, other arm. Created by Biorender.
Mice weight and temperature were recorded daily immediately before the injection during the 7 days of LPS administration. Temperature was measured using a DT-8826 (SCC Inc., Gampaha, Sri Lanka) non-contact infrared thermometer following the device procedure and protocol.
Locomotor activity was quantified for 10 min with the open field test. Each
mouse was placed gently into a rectangular field (45
The NORT is a widely applied method for assessing short-term recognition memory
based on the exploratory behavior of rodents . Here, it was used to examine
the effect of LPS on short-term visual recognition. The protocol was performed as
described previously with slight modifications. The task was carried out over a
period of 2 days: day 1 for habituation, and day 2 for testing. During the
habituation session, mice were allowed to move freely and habituated to the arena
for 10 min. After 24 h, animals underwent the familiarization phase in which each
mouse were exposed to two identical objects for 3 min (Fig. 1B). After a
retention interval of 10 min, in their home cage, the mice were returned to the
arena for the test phase, in which one of the familiar objects was replaced with
a cleanable and unmovable novel object that differed in shape, color and texture
from the familiar one (Fig. 1B). After each trial, the arena was cleaned with
10% ethanol to eliminate odor cues. In both the familiarization and test phases
of the test, the frequency of sniffing of each object was calculated (to ensure
that all mice had a similar chance of investigating both objects) as follows:
Frequency of sniffing (%) = (novel or familiar object frequency of
sniffing/total frequency of sniffing of the two objects)
The NADT was used to examine spatial recognition memory . In brief, the three arms were randomly marked as starting arm (SA), novel arm (NA), and the other arm (OA). The protocol consisted of two 3 min training and test trials. In the first trial, mice were placed in the SA, and the NA was blocked. Mice were freely allowed to move only between the SA and the OA (Fig. 1C). After 10 min, the second trial was implemented in which the NA was opened and the mice were allowed again to explore all three arms (Fig. 1C). The duration in the NA arm was calculated as an indicator of spatial recognition memory performance. Mice with intact spatial recognition memory were expected to spend more time exploring the NA.
On the last day of the study, mice were anesthetized by isoflurane inhalation.
Brains were removed and post-fixed for 48 h in 10% paraformaldehyde fixative.
For histological study, sagittal sections were cut and processed as paraffin
sections of 2–5
All data are expressed as mean
During the 7 days of LPS administration, two-way repeated measures ANOVA of body
weight showed no significant difference for days
Effect of lipopolysaccharide (LPS) on (A) body weight (g) (B)
body weight gaining (%) and (C) body temperature. Body weight were measured
daily during the 7 days of LPS administration (A). The total body weight gain
(%) at the end of LPS administration is calculated in (B). Temperature was
measured daily during the 7 days of LPS administration (C). Mice body weight and
temperature showed no significant difference compared to the control group. Data
are presented as mean
LPS-administered mice traveled a shorter distance compared with the control group, but the difference was not significant (p = 0.1413) (Fig. 3A). Furthermore, there was no significant difference in velocity (p = 0.6451) (Fig. 3B). These data were automatically calculated by EthoVision Tracking system (Fig. 3C).
Locomotor activity assessment in the open field test (OFT). (A)
No significant differences were observed in the TDM among all groups. (B) There
were no significant differences in velocity among the groups. (C) Representative
track sheets for the open field test. Data are presented as mean
During the familiarization phase, there was no significant difference in the
frequency of sniffing (%) of each object (familiar vs. novel) among the groups,
except the LPS 0.5 mg/kg group (control, p = 0.9660; LPS 0.25 mg/kg,
Frequency of sniffing (%) (A, B) and discrimination index (DI)
(C). (A) Among all groups, no significant differences were found during the
familiarization stage in the frequency of sniffing (%) of the two identical
objects, except for the LPS 0.50 mg/kg group. (B) In the test phase, a
significant difference was found in the frequency of sniffing (%) of each object
(familiar vs. novel) among the Control, LPS 0.25 mg/kg and LPS 0.50 mg/kg groups,
but not the LPS 0.75 mg/kg group. (C) There was no significant difference in the
DI in the LPS 0.25 and 0.50 mg/kg groups compared with the control group.
However, there was a significant decrease in the DI in the LPS 0.75 mg/kg group
compared with the control group. (D) Representative track sheets in the NORT
test. Data are presented as mean
During the second trial in the NADT, mice in the control group, but not the LPS groups, spent most of their time in the NA (LPS 0.25 mg/kg, p = 0.0004; LPS 0.50 mg/kg, p = 0.0012; LPS 0.75 mg/kg, p = 0.0003) (Fig. 5).
Novel arm discrimination task. Significant differences were
observed in the duration in the novel arm among all the groups compared with the
control group. Data are presented as mean
In the control group, light microscopic examination revealed normal prefrontal tissue without any sign of pathological change. The control sections contained pyramidal cells with a pale vesicular nucleus and a distinct nucleolus, in addition to normal glial cells (Fig. 6A,B). In contrast, PFC sections from mice in the LPS 0.25 mg/kg group contained an increased number of darker degenerating neurons, compared with sections from control animals. In addition, the sections contained pyramidal cells with a dark nucleus and a prominent nucleolus. Furthermore, basophilic irregular neuronal cells devoid of distinct nuclei (necrotic cells) were detected. Glial cells were also observed (Fig. 6C,D). Sections from the LPS 0.5 mg/kg group contained a higher number of pyramidal cells with a pale vesicular nucleus and a prominent nucleolus compared with the control group. Pyramidal cells with dark vesicular nuclei and multiple processes were also detected. Additionally, some basophilic cells were surrounded by empty spaces (apoptotic cells) (Fig. 6E,F). In comparison, sections from the LPS 0.75 mg/kg group contained an increased number of darker neurons and fewer pyramidal cells with a pale vesicular nucleus an obvious nucleolus. Moreover, these sections contained a larger number of degenerating dark basophilic cells, compared with the control group (Fig. 6G,H).
Representative photomicrographs of H&E-stained
sections of the prefrontal cortex (PFC) from mice in the different groups. (A,B)
Control group. (A) Normal cortical delineation and cellular distribution of
neurons and neuropil in the molecular layer (ML), external granular layer (EGL),
external pyramidal layer (EPL), internal granular layer (IGL), internal pyramidal
layer (IPL) and polymorphic layer (PL). (B) Pyramidal cells (PC) with a pale
vesicular nucleus and a distinct nucleolus. Normal-appearing granular cells (GC),
microglial cells (Mg) and blood capillaries (C) were also detected. (C,D) LPS
(0.25 mg/kg) group. (C) Poor cortical delineation with increased darker neurons
compared with the control tissue. (D) Pyramidal cells (PC) appeared condensed
with dark nuclei. The capillaries were more congested with red blood cells
compared with the control. The sections also showed more microglial cells and
congested capillaries (C). (E,F) LPS (0.5 mg/kg) group. (E) Disorganized
delineation of the cortical layers and increased darker neurons compared with
control tissue. (F) The pyramidal cells appeared more condensed with dark
cytoplasm. Furthermore, the neuropil appeared vacuolated with the presence of
congested capillaries (C). (G,H) LPS (0.75 mg/kg) group. (G) Disrupted
delineation of cortical layers compared with the control group. (H) Multiple
condensed and dark pyramidal cells. These sections also showed more microglial
cells (Mg) and congested capillaries (C). (A, C, E, G:
In this study, Swiss mice were used to
investigate the dose-dependent PFC-related recognition memory deficits produced
by chronic peripheral LPS administration. Neuroinflammation caused by chronic
microglial hyperactivation has been identified as a leading cause of cognitive
impairment and neurodegenerative disease . By releasing numerous
proinflammatory cytokines, hyperactivated microglia substantially increase brain
neuroinflammatory and neurotoxic processes . Elevation of proinflammatory
cytokines such as tumor necrosis factor-
LPS may lead to multiple non-specific behavioral effects, and can reduce activity, social interaction and exploratory behavior in mice . Thus, the OFT was performed, and body weight and temperature were recorded to evaluate general health and locomotion. Mice receiving LPS showed no significant differences in weight gain, temperature or locomotor activity during the course of LPS administration. Therefore, there was no interaction of these parameters with memory parameters .
Recognition memory is generally defined as the ability to judge whether an item
or event is previously experienced . Damage to the PFC has been found to
affect recognition memory tasks . The NORT is a behavioral task commonly used
to evaluate visual object recognition memory in rodents, including PFC-dependent
recognition memory [37, 38]. Mice with normal PFC functions prefer to investigate
the novel object rather than the familiar object, whereas mice with a damaged PFC
are predicted to have no preference . Our NORT results demonstrate
recognition memory impairment in mice administered 0.75 mg/kg LPS. In particular,
analysis of the frequency of sniffing (%) showed that the LPS 0.75 mg/kg group
had no significant preference for either object, indicating a recognition memory
deficit. To the best of our knowledge, this is the first study to demonstrate
that LPS at a dose of 0.75 mg/kg causes PFC-related memory impairment and
histological alterations. The LPS groups given the 0.25 and 0.5 mg/kg doses
exhibited performance similar to the control group (i.e., a preference for the
novel object over the familiar object). In line with our results, a previous
study showed that adult male Swiss albino mice explored the novel object markedly
more than the familiar object after 3 days of LPS 0.25 mg/kg injections . In
comparison, 7 days of LPS 0.75 mg/kg administration significantly impairs the
ability to discriminate the novel and familiar objects . In line with the
previous study, we found here that LPS 0.75 mg/kg caused a significant decrease
in the DI. While mice in the LPS 0.25 and 0.50 mg/kg groups showed reduced novel
object discrimination, the differences were not significant. There is a lack of
concordance in the literature on the effects of acute and chronic administration
of 0.25 and 0.5 mg/kg LPS on recognition memory performance. For example,
Czerniawski et al.  showed that acute LPS administration, by
either i.p. or intracerebroventricular (i.c.v.) administration, impairs novel
object recognition. In male Wistar rats injected with a single dose of LPS 0.50
mg/kg, there is a significant decrease in the ability to discriminate between the
novel and familiar objects . Moreover, several reports found that chronic
administration of LPS 0.25 mg/kg for 7 days reduced discrimination in the novel
object recognition task [8, 24, 41]. Feng et al.  reported that
a single peripheral injection of
Here, we also assessed whether chronic peripheral LPS administration affected another type of recognition memory—spatial recognition. NADT is a simple two-trial hippocampus-dependent task commonly used to assess spatial recognition memory based on rodents’ innate tendency to investigate a new environment [45, 46]. Our findings revealed that compared with the control group, all LPS groups significantly spent a shorter duration in the NA, reflecting a spatial recognition memory deficit. LPS administration resulted in no preference for the new arm throughout the exploration time.
To link the PFC neurodegeneration with the cognitive deficits, histological examination was conducted using H&E staining. In general, PFC neurodegeneration was observed in all sections from LPS-treated mice. However, each dose produced different degrees of degeneration. In particular, mice given the 0.75 mg/kg dose showed more dark neurons compared with the control group. In agreement with the behavioral results, the 0.75 mg/kg dose had the greatest impairment effect.
Taken together, our results demonstrate that the peripheral administration of 0.75 mg/kg LPS impaired both object and spatial recognition memory, while the 0.25 and 0.50 mg/kg doses significantly impaired spatial recognition memory only. Thus, we hypothesize that recognition memory deficits following chronic administration of 0.25 and 0.50 mg/kg LPS are likely to be associated mainly with hippocampal insult, whereas the 0.75 mg/kg dose impacts both the hippocampus and prefrontal cortex. Indeed, the 0.25 and 0.50 mg/kg groups had a frequency of sniffing (%) similar to the control group, indicating that a greater dose of LPS is needed to induce PFC-related recognition memory deficits. To the best of our knowledge, this is the first study linking the chronic administration of LPS at 0.50 and 0.75 mg/kg doses with PFC-related recognition memory impairment in mice.
In summary, we investigated the effects of administration of different doses of LPS on recognition memory and PFC histological changes in mice. Results of behavioral assessment and histopathological examination show that chronic peripheral administration of LPS at 0.75 mg/kg is the optimal dose for studying neuroinflammation-associated recognition memory deficits. Further study by immunostaining is needed to evaluate the effect of LPS on different neuronal and glial cells. Furthermore, electrophysiological investigations are required to clarify the contributions of the PFC and hippocampus to the recognition memory impairment induced by peripheral LPS administration.
AD, Alzheimer disease; LPS, lipopolysaccharide; NORT, novel object recognition test; NDAT, novel arm discrimination test; OFT, open field; PFC, prefrontal cortex; NOR, novel object recognition; IP, aspartate transaminase; ICV, total bilirubin; mPFC, medial prefrontal cortex.
NAA, KAB, RAM and BSA designed the research study. NAA performed the research. KAB helped carried out the experiments. NAA analyzed the data and wrote the manuscript. RAM and BSA supervised the project. HMA provided critical feedback and helped shape the research. RAM, BSA and HMA contributed to editorial changes in the final manuscript. All authors read and approved the final manuscript.
All mouse experiments followed the protocols and guidelines of the Animal Unit Department at King Fahad Medical Research Center, Jeddah, SA. The study was approved by the Unit of Biomedical Ethics, Faculty of Medicine, King Abdul-Aziz University, Jeddah, SA (Reference No. 603-20), which complied with the “System of Ethics of Research on Living Creatures” guidelines prepared by King Abdulaziz City for Science and Technology, and were approved by Royal Decree No. M/59 dated 24/08/2010.
We would like to thank Rahaf Al dhahri, Khlood Alyami, and Ghada Alshahrani for their assistance in manuscript revising and figures designing and we would like to extend our special thanks to Gamal S.Abd El-Aziz for his help in the histological examination.
This research received no external funding.
The author declares no conflict of interest.