1. INTRODUCTION:

According to the World Health Organization (WHO), 3.8% of the world's population, or more than 350 million individuals, currently suffer from depression¹. Various stressful events can be considered the most effective in modelling depressive behavior². Both patients and doctors frequently fail to detect depression's subtler signs³. Laboratory animals peripheral innate immune systems are acutely activated by the introduction of LPS, which triggers the Toll-like receptor 4 transmembrane cytokine pathway⁴.

Increased immobility in the forced swim test and tail suspension test, a reduction in sweetened solution consumption, and a suppression of sexual behaviour are all signs that LPS causes depressive-like behaviour⁵. The dysfunctional of hypothalamic-pituitary-adrenal (HPA) axis and aberrant cytokine release is main cause of depression⁶. LPS-induced depression and prolonged, unpredictable moderate stress in rats are more sensitive and appropriate than the normal mouse FST⁷. One of the risk factors for Alzheimer's disease is depression⁸.

Ketamine (Ket), which belongs to the family of phencyclidine (N-1-phenycyclohexypiperidine, PCP) binds to the phencyclidine site to generate anaesthesia, is a non-competitive N-methyl-D-aspartate receptor (NMDA) receptor antagonist⁹. Additionally, because depression is regarded to be a condition linked to stress and has a high morbidity rate, there is increased interest in using the anaesthetic drug Ket for the treatment of the illness.

An earlier study demonstrated that Ket has an anti-inflammatory impact during sepsis patients surgery ¹⁰. In many people, it can effectively reduce depression symptoms and suicidal thoughts¹¹. The chronic mild stress and social defeat stress models both benefit from the prolonged antidepressant effects of Ket and R-ketamine¹². Compared to (R)-Ket, (S)-Ket (esketamine) has an approximately 4-fold higher affinity for the NMDA receptor. Esketamine administered intranasally and claimed to have antidepressant effects was developed by Janssen Pharmaceutical¹³. The typical negative effects of traditional antidepressants are numerous.¹⁴ As Ket at 90–100mpk, intraperitoneal injection is lethal and used to euthanize the mice¹⁵, therefore, we attempted to evaluate with sub anesthetic dose of Ket in the sub-acute LPS induced depression like phenotype in mice.

Since B/W is widely seen as a good indicator of wellbeing, we attempted to create a depression condition with an adjustment to the dose and duration of LPS, where there was a non-significant drop in B/W of animals.

All the experiments were performed by male experimenter, as the sex of the human experimenter influences the way mice respond to Ket, a drug used to treat depression and anxiety in humans¹⁶.

Microglia function by triggering an inflammatory response to defend the body against any potentially hazardous stimuli. Cytokines are crucial for the polarisation process that causes microglia to enter the M1 active state¹⁷. As majority of studies restricted their interest to the hippocampal area¹⁸, especially to CA-1 and CA3 region¹⁹, we measured the immunoreactivity cells of anti-Iba1 antibody and CD11b cells in the hippocampal region and behavioral response after peripheral sub-acute LPS injection by a male experimenter.

2. Materials and Methods:

2.1 Animals:

Male C57BL/6 mice weighing (20 to 25g) were purchased from a single source (Vivobiotech, Hyderabad) and kept in groups of four in cages with individual ventilation. Animals were provided unlimited access to autoclaved water and SDS pelleted feed. Mice were kept in animal rooms that were provided light from 8a.m. to 8p.m. and kept at a temperature between 21 to 24°C with a relative humidity of 40 to 70%. All behavioural tests were carried out between the hours of 9:00 AM and 5:00 PM. The Institutional Animal Ethics Committee of Jubilant Biosys Ltd. in Bengaluru granted approval for the animal protocol under registration number IAEC/JDC/2018/150.

2.2 Reagents:

Lipopolysaccharide (LPS, Escherichia coli, serotype O111:B4, L2630, Lot# 028M4022V, Sigma), Ketamine (Lot # B5B0394, Baxter), RIPA buffer (9806, Cell Signaling Technology), Sucrose (Lot # 57-50-1, Fisher Scientific). Primary antibodies rabbit anti-mouse CD11b poly clonal antibody (Abcam: ab75476, Lot # GR195378-2); Rat anti-mouse Iba-1 monoclonal antibody (Abcam: (ab283346, Lot # GR3419831-3) and secondary specific to the primary host viz. Goat anti-rabbit polyclonal antibody conjugated to Alexa fluor 488 (Abcam: A11034, Lot # 2286890), Goat anti-rat polyclonal antibody conjugated to Alexa Fluor 594 (Abcam: ab150160, Lot # GR3371713-6) were used. All other reagents were used from Sigma Chemical Co.

2.3 Experimental Groups:

The group allocation were mentioned in Table 1. Half of the animals from each group underwent behavioral tests (SPT, FST) and the other half used for immunofluorescence (IF) analysis. The mice were administered multiple i.p. injection of Ket or equal volume of physiological saline to corresponding groups for consecutive 6 days at 1 h post LPS injection.

Table 1: Effect of Ket on relative spleen ratio, relative thymus ratio and relative adrenal ratio of sub-acute LPS injected C57Bl/6 mice.

Groups	Relative spleen ratio Avg ± SEM	Relative thymus ratio Avg ± SEM	Relative adrenal ratio Avg±SEM
PBS + Saline	0.00245 ± 0.0002	0.00167 ± 0.0001	0.00035 ± 0.00002
LPS @ 300µg/kg + Saline	0.00716 ± 0.0007 ^$$$	0.00097 ± 0.0001 ^$$	0.00045 ± 0.00004
LPS @ 300µg/kg + Ket @ 3mpk	0.00643 ± 0.0004	0.00102 ± 0.0001	0.00041 ± 0.00004
LPS @ 300µg/kg + Ket @ 10mpk	0.00624 ± 0.0003	0.00132 ± 0.00006	0.00038 ± 0.00002
LPS @ 300µg/kg + Ket @ 30mpk	0.00917 ± 0.001	0.00092 ± 0.00008	0.00045 ± 0.00003

$$p＜0.01 and $$$p＜0.001 vs. Saline; One-way ANOVA followed by Dunnett’s post hoc test.

2.4 Behavioral experiments:

2.4.1 Mouse Sucrose Preference Test:

The procedure started with an adaptation period on Day 5, where mice were exposed to sucrose solution with two bottles of 1% (w/v) sucrose solution placed in each cage. On Day 6, sucrose solution in one bottle was replaced with normal autoclaved water during 24h. Mice were deprived of water and food for further 6h, post this adaptation period. The test was performed on Day 7 after last saline or Ket injection to evaluate anhedonia. Mice were given a free choice between two bottles containing 100mL of 1% w/v sucrose solution and 100 ml of autoclaved water for the sucrose preference test. The bottles were weighed at the start and one hour during the trial, and the amounts of water and sucrose solution consumed were noted. The % sucrose preference was reported.

2.4.2 Mouse Forced Swim Test:

In FST, mice are forced to swim in a restricted space from which they cannot escape, and are induced to a characteristic behavior of immobility²⁰. Briefly stated, each mouse was lowered into a glass cylinder with a diameter of 23cm and a height of 30cm that contained 14cm of water that was kept at a temperature of 24± 1°C. Between each testing session, the water was changed. The mice were kept in the cylinder for 6 minutes before being put back in their cage. Over the final five minutes of the test, the immobility time was calculated. When mice floated passively in the water, moving only enough to keep their heads above the water's surface, that condition was referred to as immobility⁷.

2.4.3 Tissues collected at the time of necropsy for analysis:

On Day 7, after the experimental regimen (1 h following saline/Ket injection), half of the mice were slightly anesthetized under CO₂ asphyxia. The whole brain was extracted from the animal after it had died from CO₂ asphyxia. Half of it was immediately snap frozen in liquid nitrogen and stored at -80°C until exposure analysis and remaining half brain was dipped in freshly prepared ice-cold 4% paraformaldehyde fixative prepared in phosphate-buffered saline (PBS) buffer.

2.4.4 Immunofluorescence (IF) Staining of brain tissue:

Post fixation, tissues were embedded with optimal cutting temperature (OCT) media and freezed on powdered dry ice. These tissues were then allowed to equilibrate inside the cryotome, cut into 14µm thick slices, mounted on poly-L-lysine coated slides. These slides were processed further with slight modification to CSH protocol. For imaging, Leica DMi8 inverted fluorescence microscope was used and images were quantified for the phenotypic changes of microglia and CD11b cells from the hippocampus region of mice.

2.4.5 Statistical Analysis:

Data were reported as mean±SEM of 4-8 animals. Statistical analysis was performed using Graph pad prism (version 9.0.0 GraphPad Software, San Diego, CA, USA). Comparisons of all study parameters were made between the treatment groups and respective vehicle control groups by One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. A p value less than 0.05 was considered significant.

3. RESULTS:

3.1% change in B/W:

Due to the injection of LPS, there was a considerable decline in B/W in the LPS group on Days 3 and 5, which gradually recovered on Day 7 (From in-house result, data not shown here)

On Days 3 and 5, Ket at 3mpk shown a considerable improvement in B/W. On the same days, the Ket treatment (10mpk) exhibited a non-significant but notable improvement, while the Ket treatment (30mpk) did not. As a result, Day 7 was chosen as the day for all experiments.

3.2 Effect of Ket on LPS induced SPT in male C57Bl/6 mice:

The decrease in preference for sucrose solution over water confirms the depressive-like behaviour that measures anhedonia. The desire for sucrose in mice decreased significantly (p<0.05) following several LPS injections. The saline-injected animals showed a 72% preference for water over a sucrose 1% solution. When compared to the LPS group, Ket administration at 30 mpk increased the preference for sucrose (65.6±1.1 Vs 34.9±7.2, Fig. 1A).

3.3 Effect of Ket on LPS induced FST in male C57Bl/6 mice:

When sub-acute LPS was injected into mice, the LPS group experienced considerably (p<0.0001) longer periods of immobility than the control group (148.4±2.1 Vs 74±5.1). When compared to the LPS group, Ket therapy at 10 mpk minimize the increase in immobility time (148.4±2.1 Vs 131±2.5) and at 30 mpk prevented the increase in immobility duration (148.4±2.1 Vs 85.5 ±2.4, Fig. 1B).

[A]

[B]

[C]

Figure 1: % SPT [A], FST [B], Intensity of Iba1 cells activation [C]. $ p<0.05, $$$ p<0.001, $$$$ p<0.0001 when LPS group compared with Saline group, *p<0.05, **p<0.01 and **** p<0.0001 when Ket dosed groups compared with LPS group.

3.4 Effect of Ket on LPS induced relative spleen, thymus and adrenal ratio in male C57Bl/6 mice:

When compared to the control group, mice that received sub acute LPS injections experienced substantial (p<0.001) splenomegaly (Table 1) and a significant (p<0.01) decline in the thymus to B/W ratio (Table 1). Both metrics did not significantly improve after Ket administration.

In the LPS group, there was a marginal rise in the adrenal to B/W ratio (Table 1). With the three Ket dosages that were evaluated, there was no discernible improvement in the adrenal to B/W ratio.

3.5 Effect of Ket on LPS induced intensity of Iba1, number of microglia and intensity of CD11b cells in hippocampus region of male C57Bl/6 mice:

The Iba1 intensity in the hippocampal region showed a significant modulation in the LPS group (Table 2). With Ket at 30mpk dose, there was significant change in the activation related intensity of Iba1.

The number of microglia seen in the hippocampal region increased non-significantly in mice following subacute LPS challenges (Table 2).

The CD11b cells excitation in the hippocampal region showed a non-significant modulation in the LPS group (Table 2). With three Ket test dosages, there was no discernible change in the aforementioned metrics.

Table 2: Effect of Ket on Iba-1 intensity, number of microglia and CD11b+ intensity in the hippocampus region of sub-acute LPS injected C57Bl/6 mice.

Groups

Intensity Iba1

Avg ±SEM

Number of microglia

Avg ±SEM

Intensity CD11b⁺

Avg±SEM

PBS+Saline

0.026 ± 0.004

38.3±

7.8

0.76± 0.02

LPS @ 300µg/kg + Saline

0.068 ±

0.002 ^$$$

66.1±

0.3

0.69±

0.02

KET @3 mpk+

LPS @ 300µg/kg

0.070 ±

0.001

70.2±

2.4

0.68±

0.02

KET @10 mpk+ LPS @ 300µg/kg

0.060 ±

0.001

60.4±4.9

0.65±

0.00

KET @30 mpk+ LPS @ 300µg/kg

0.045 ± 0.004 **

57.0±8.9

0.69± 0.02

$$$p＜0.001 vs. Saline; ** p<0.01 when Ket dosed groups compared with LPS group. One-way ANOVA followed by Dunnett’s post hoc test.

4. DISCUSSION:

The primary goal of this study was to determine whether mice may exhibit depressant-like behaviour after receiving sub-acute LPS injection. This was demonstrated by longer immobility times in the FST and decreased preference for a sucrose solution²¹. Mice exposed to LPS exhibited illness behaviour and lost body weight. Because B/W is widely seen as a good indicator of wellbeing, we attempted to create a depressed condition with a small adjustment to the LPS dose²². And after 6 days of LPS injection (300µg/kg), a depressant-like animal model was noticed in which B/W significantly decreased until Day 5 and then began to recover by Day 7.

Female mice were not used as timing in the oestrus cycle, can also modulate the murine response to an immune challenge. At 24 and 48hours following LPS, there was an increase in Iba-1 immunoreactivity and de-ramified microglia and antidepressants drug can modulate it. IHC data with Iba-1 showed that the hippocampus had activated microglia 2 days after the LPS challenge and that CD11b expression had risen there for 3 months after infection¹⁸. Hippocampal regions are more vulnerable to LPS stress which cause depression²³, Our IF result with Iba-1 intensity level revealed activated microglia in the hippocampus 6 days after LPS challenge and Ket able to reverse the degree of intensity. Iba1-reactive cell numbers were meaningfully higher whereas the treatment of Ket did not lower this number. CD11b expression maintain normal level in the hippocampus area, possibly, age of animal, dose, duration and serotype of LPS are important factor for determining the expression level.

We chose Ket over other anti-depressants because it treated pain that was measured by filaments and cold hypersensitivities stimuli at higher dosages and reduced spontaneous pain at low levels. Ket at 10mpk has been shown to treat depression without having any negative psychomotor effects¹², as antidepressants do not increase general motor activity²⁴.

The stress model was chosen since this LPS exposure tends to the onset of thymus atrophy symptoms, an increase in relative spleen weight, and hypertrophy of adrenal gland²⁵. However, it is probable that with this experimental paradigm, the Ket dose used was insufficient to change the relative thymic weight, spleen weight and adrenal weight.

Day 7 exposure in the brain and plasma of male C57BL/6 mice was examined 1 hour after Ket injection, and the Brain to plasma (B/P) ratio for 10mpk and 30 mpk was determined to be 0.70 and 0.82, respectively. It is necessary to conduct further in-depth research on Ket's long-lasting antidepressant effects at earlier time periods with different serotype of LPS.

5. CONCLUSION:

Our findings supported the hypothesis that this LPS serotype generates depression-like symptoms in mice after a 6-day injection (sub-acute regimen), altering behavioural measures including FST and significantly decreasing sucrose preference. When compared to the LPS group on Day 7, daily Ket treatment in our rodent model improved antidepressant-like behavioural distress (i.e., decreased immobility time in FST and increased sucrose preference).

In this experimental paradigm, LPS induces a significant immunological reaction in the brain that results in the activation of microglia and Ket was able to change the Iba-1 intensity.

Pharmacological responses are driven by the total concentration of a drug at the site of action, such as the brain. In addition, Ket demonstrated antidepressant-like effect, and showed a correlation between phenotypic changes of microglia and having good B/P ratio at 10 mpk and 30 mpk. To our knowledge, this is the more sensitive, appropriate and first report of Ket on LPS induced depression. These results divulge new insights to the neuroinflammation inhibition.

6. ACKNOWLEDGEMENTS:

We would like to thank all Biology scientists of Jubilant Biosys Ltd, Bengaluru.

7. CONFLICT OF INTEREST:

The other authors declare no conflict of interest.

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Received on 15.11.2022 Modified on 19.04.2023

Research J. Pharm. and Tech 2024; 17(4):1585-1589.

DOI: 10.52711/0974-360X.2024.00250