Experimental design

56 male, healthy Swiss albino mice (C57BL/6 strain) were between 6 and 8 weeks old and weighed 20 to 30 grams. The animals were sourced from the ECF in Egypt. Mice were thoroughly inspected for microbiological or parasitic infections and kept in plastic cages at 21°C and 60% humidity. Keeping mice in the usual pathogen-free conditions. They were fed a conventional laboratory meal consisting of 24% protein, 4% fat, 4–5% fiber, and water ad-libitum. The animals were fed, housed, and handled in accordance with the Guidelines for the Care and Use of Laboratory Animals.

Three studies were carried out, each in a separate group of mice. Our study duration is four weeks. Briefly, mice were allocated into 3 groups in addition to the control group (n=8), which feeds normally without exposure to any stressors. Group 1: (sleep deprivation experiment) Mice (n=16) received 20 hours of sleep deprivation utilizing a gently revolving wheel (1.0 RPM x 20 hours/day), followed by a 4-hour sleep opportunity in their home cage. The sleep deprivation wheels are plastic rotating wheels with a 9.0-inch diameter. Each one is held together by a sturdy steel plate on one side and a transparent plastic sheet on the other. The wheel cages are intended to facilitate simultaneous spinning of the wheels as well as free access to food and water. The wheel speed was kept constant at 1.0 rotations per minute. Group 2: (noise-exposure experiment) mice (n=16) placed into cages supported by an electrostatic speaker. The white continuous noise was generated using a noise generator supported by a microphone placed at the test mice location for noise output for 12 hours during the experiment daytime. Group 3: (high fat diet experiment) mice (n=16) were continuing to feed with high density (60 kcal% fat diet) for four weeks. More food is usually added as needed. At the end of each week, 4 mice were sacrificed from each group to collect plasma and subjected to CRP evaluation.

CRP level measurement

The blood samples were collected using EDTA tubes at the end of each week from four mice for four weeks from each experimental group, then centrifuged each sample for 15 min at 3000 rpm at 2~8°C within 30 min of collection.  Quantitative measurement of C Reactive Protein (CRP) level in plasma was estimated by the Mouse C Reactive Protein (CRP) ELISA 96-well plate kit (ab222511).

Statistical analysis

Depending on the data distribution, continuous variables are displayed as the median or mean ± SD. The statistical significance of the differences between groups was determined using the Student t-test, with the threshold of significance set at p < 0.05.

Fifty-six (56) male, healthy Swiss albino mice allocated into 3 test groups in addition to the control group. Each group has 8 mice. Our study extended for four weeks. At the end of each week, 4 mice were sacrificed from each experimental group to collect plasma using EDTA tubes and subjected to CRP quantitative evaluation using the Mouse C Reactive Protein (CRP) ELISA 96-well plate kit with plate reader.

A significant increase in plasma CRP level was observed in our stressed experimental groups, as shown in Table 1. CRP levels were found to be steadily increasing after two weeks, and gradually elevated in plasma with prolonged time of sleep deprivation in group 1 (sleep deprivation experiment), which is higher than control group mice and most significant after the third and fourth weeks of experimentation procedures (33mg/l ±6, 56mg/l ± 3) (p < 0.001) (Fig., 1).

Noise stress becomes more significant as exposure increases throughout the experiment time on the CRP plasma level. The fourth week was the most significant on the noise experiment (71 mg/l ± 4, p < 0.001) (Fig., 2). Moreover, the results strongly supported that the plasma CRP levels were exceptionally increased in the noise group by time more than in other groups (groups 1 & 3) from the first week to the end of our experiment.  Plasma CRP level was also slightly elevated in (group 3) that fed on a high fat diet for four weeks (60 mg/l ± 6,  p < 0.005) compared with the control group (50 mg/l ± 6) (Fig.,3).

Figures & Tables

Discussion

C-reactive protein (CRP), identified as a protein that precipitates Streptococcus pneumoniae C-polysaccharide, has long been recognized as a sensitive indicator of inflammation [12]. CRP has several pro- and anti-inflammatory actions. Although not unique to a particular disease process, CRP is routinely employed as a static assessment, and CRP levels have been linked to a variety of diseases [13].

Despite substantial studies, the effects of sleep deprivation on C-reactive protein (CRP), are inconclusive, whereas other studies found no difference in CRP levels [14]. Furthermore, studies found decreases in CRP levels [15], whereas others found increases in CRP levels [16.17] which is consistent with our findings. The relation between CRP level and noise is extensively documented in different studies. C-reactive protein was significantly reduced in the noise exposed rats’ experiment [18], while its concentrations have been reported to rise after prolonged noise stress exposure [19]. Our study linked elevated CRP levels to noise stress.

There is an association between fatty acid consumption and CRP inflammatory indicators. Glial cells are activated by a high-fat diet, causing them to generate and release cytokines and C-reactive protein as inflammatory mediators [20]. Our study aims to assess if dietary fatty acid consumption corresponds with serum high-sensitivity C-reactive protein levels. Consistent with our findings, epidemiological studies linked chronic stress resulting from a high fat diet with inflammatory markers [21, 22]. Murakami et al., [23] found no significant link between a high fat diet and elevated CRP. Whereas other study found no significant link between a high fat diet and a high CRP level [24]. Therefore, a high intake of meals reached with fat could increase inflammation, as reflected in CRP level.

Our studies directly implicate the stress induced increase the level of CRP. Its level increased in mice exposed to stressful events over a long period of time, stressors of sleep deprivation, noise, as well as high fat diet intake, in accordance with previous epidemiological research. Collectively, these findings suggest that alterations in CRP explain the increased inflammation risk linked to our experimental designed stressors.

In conclusion, our findings support the concept that some anxiety and stress-related disorders are associated with elevated levels of inflammatory markers like CRP. The relationship between CRP and sleep deprivation and noise, as well as high fat diet intake, was more likely to show elevated plasma CRP levels.  More research is needed to determine the potential causative and mechanistic ties involved in the triggering of CRP with stressors, as well as to correlate external stresses with other subclinical inflammatory indicators.

Conflict of Interest

The author declare that there is no conflict of interest regarding the publication of this paper.

References