Introduction
Despite the new methods of pharmacotherapy of periodontitis, incidence of chronic periodontitis is still high. According to various data, incidence of this disease in people aged 40–50 years is 35–50%, in elderly people — 90% [1, 2]. Chronic periodontitis not only worsens the quality of life, but also contributes to somatic diseases [3]. In turn, various forms of somatic pathology influence development of periodontitis.
Chronic pain syndrome is one of the most common diseases in middle-aged and older people (33–50%) [4]. Considering the prevalence and high social significance of these diseases, as well as the need to develop an individual treatment approach, analysis of pathogenesis of periodontitis in patients with chronic pain is still an urgent problem. The previously established changes in release of pro-inflammatory and anti-inflammatory cytokines in pain syndromes, as well as weak antinociceptive effect of the leading periodontal pathogen (Porphyromonas gingivalis) require research of effect of chronic pain syndrome on the course of periodontitis [5, 6].
It is known that neutrophils and macrophages are essential in development of inflammation. They realize a phagocytic function and secrete various biologically active substances regulating various phases of inflammation [7, 8], including pro-inflammatory and anti-inflammatory interleukins, growth factors, and metalloproteinases. At the same time, phagocytic barrier is important for not only resistance of periodontium to pathogenic microflora, but also regenerative processes accompanying inflammation. It is known that bone tissue and periodontal ligaments are characterized by low reparative potential that results slow and usually incomplete regeneration of these structures [9].
Dysfunction of neutrophils and macrophages has been previously established in experimental periodontitis [10]. However, there are no literature data on changes in functional activity of macrophages and neutrophils in patients with periodontitis and chronic pain syndrome.
The purpose of the study was to study phagocytic activity of neutrophils and adhesive ability of macrophages in rats with experimental periodontitis and chronic pain syndrome.
Material and methods
The study was performed on 128 male Wistar rats weighing 180–220 g. Laboratory animals were kept in standard conditions with free access to food and water. The studies were performed in compliance with the WMA Declaration of Helsinki on Animal Use in Biomedical Research (2000), Directive of the European Community (86/609EC), Rules of Good Laboratory Practice in the Russian Federation (order of the Ministry of Health No. 199n dated 04/01/2016) and Interstate standards GOST 33215-2014, GOST 33216-2014 "Guidelines for the maintenance and care of laboratory animals". The regional ethics committee approved the study (protocol No. 2 dated 10/19/2020).
We distinguished the following groups of animals: intact — 8 rats; modeling of chronic pain syndrome — 24 rats; false-operated animals — 24 rats; modeling of experimental periodontitis — 24 rats; modeling of experimental periodontitis in false-operated animals — 24 rats; modeling of experimental periodontitis in animals with chronic pain syndrome — 24 rats.
Experimental periodontitis was modeled using the method proposed by Volozhin A.I. and Vinogradova S.I. (1990) [11]. Under chloral hydrate anesthesia (400 mg/kg intraperitoneally), a silk thread in the form of a figure-eight was applied to incisors of the lower jaw with subsequent immersion of ligature into periodontal groove and its fixation with additional knots. The thread was left for 14 days and then removed. Rats were taken out of the experiment after 7, 14 and 21 days after removal of thread by overdose of anesthetic drug.
Chronic pain syndrome was modeled using the above-described method [12]. Bilateral ligation of sciatic nerves within the middle third of the thigh was performed under chloral hydrate anesthesia. After 2 weeks, surgical wound healed, and the animals were used in further experiments. False-operated animals underwent a similar surgery without ligation of sciatic nerves. False-operated rats and animals with chronic pain were withdrawn from the experiment in 3, 5, 6 and 7 weeks after surgery. These periods were determined by the protocol of experimental study of periodontitis in rats with chronic pain syndrome. In rats with chronic pain syndrome and false-operated animals, experimental periodontitis was modeled in 2 weeks after appropriate surgery.
Phagocytic activity of neutrophils was analyzed using conventional method. We used the following parameters: phagocytic index (number of actively phagocytic neutrophils per 100 cells), phagocytic number (mean number of microbial bodies captured by 1 phagocytic neutrophil), opsonophagocytic index (mean number of absorbed microbial bodies per 100 neutrophils) [10].
Adhesive properties of macrophages characterizing their functional activity were studied using traditional method (adhesion of macrophages to a glass surface in Petri dishes) [10]. We analyzed the percentage of macrophages from total number of cells in smear and relative number of macrophages adhering to the glass in population.
Statistical analysis
Normality of distribution was assessed using the Shapiro–Wilk test. We also analyzed homogeneity of variances using the Levene’s test. Statistical hypotheses were tested using the non-parametric Mann–Whitney U-test. We applied non-parametric statistical methods due to small sample size, heterogeneous distribution of data and unequal variances. Data are presented as median (Me), lower (Ql) and upper (Qu) quartiles. The null hypothesis was rejected at p-value<0.05. Statistical analysis was carried out using the MS Excel and Statistica 10 software.
Results
We observed decrease of phagocytic index by 21.5% (p<0.05) and opsonophagocytic index by 21.3% (p<0.05) in 3 weeks after the end of modeling of chronic pain syndrome compared to the intact group (Table 1). Animals with experimental periodontitis were characterized by decrease of phagocytic index by 38.7% (p<0.05) and opsonophagocytic index by 32.0% (p<0.05) in 7 days after removal of thread. Phagocytic and opsonophagocytic indexes significantly increased in 21 days after modeling of experimental periodontitis. Phagocytic number was significantly higher in animals with experimental periodontitis compared to intact rats (by 60.0%, p<0.05). Animals with experimental periodontitis and chronic pain syndrome were characterized by decrease of phagocytic index by 22.8% (p<0.05) and opsonophagocytic index by 30.1% (p<0.05) in 7 days after removal of thread compared to rats with experimental periodontitis.
Table 1. Phagocytic activity of neutrophils in rats with experimental periodontitis and chronic pain syndrome
|
Variable Group |
Period of experiment |
Phagocytic index, n |
Phagocytic number, n |
Opsonophagocytic index, n |
|
Intact group |
46.5 [41.5; 49.5] |
1.5 [1.3; 1.7] |
61.0 [54.5; 79.0] |
|
|
Chronic pain syndrome |
3 weeks |
36.5 [35.0; 40.0]x |
1.4 [1.2; 1.6] |
48.0 [46.0; 59.0]x |
|
5 weeks |
44.5 [39.0; 48.0] |
1.5 [1.3; 1.5] |
60.0 [52.0; 64.0] |
|
|
6 weeks |
43.5 [39.0; 46.0] |
1.5 [1.4; 1.7] |
68.5 [65.0; 72.0] |
|
|
7 weeks |
45.0 [42.0; 49.0] |
1.6 [1.4; 1.7] |
66.5 [62.0; 81.0] |
|
|
False-operated rats |
5 weeks |
48.0 [42.0; 49.0] |
1.4 [1.3; 1.5] |
62.5 [55.0; 74.0] |
|
6 weeks |
45.5 [40.0; 50.0] |
1.6 [1.3; 1.7] |
67.5 [53.0; 73.0] |
|
|
7 weeks |
44.0 [39.0; 49.0] |
1.5 [1.3; 1.6] |
62.5 [62.0; 67.0] |
|
|
Experimental periodontitis |
7 days |
28.5 [26.0; 31.5]x |
1.5 [1.3; 1.6] |
41.5 [36.5; 48.0]x |
|
14 days |
31.0 [28.0; 32.5]x |
2.0 [1.4; 2.8] |
56.0 [49.0; 64.0] |
|
|
21 days |
44.5 [40.5; 45.0] |
2.4 [2.0; 3.0]x |
107.5 [93.0; 127.0]x |
|
|
Experimental periodontitis in false-operated rats |
7 days |
30.0 [26.5; 31.5]x |
1.5 [1.4; 1.6] |
40.0 [37.0; 49.5] |
|
14 days |
30.5 [28.0; 33.0]x |
2.5 [2.0; 2.7] |
57.0 [53.0; 67.5] |
|
|
21 days |
48.0 [44.0; 51.5] |
2.1 [1.6; 2.8]x |
95.0 [90.0; 119.5]x |
|
|
Experimental periodontitis in chronic pain syndrome |
7 days |
22.0 [20.0; 25.0]* |
1.35 [1.2; 1.4] |
29.0 [28.0; 34.0]* |
|
14 days |
29.0 [27.5; 32.5] |
1.7 [1.4; 2.1] |
44.0 [37.0; 58.5] |
|
|
21 days |
43.5 [39.0; 46.0] |
2.3 [2.2; 2.6] |
99.0 [84.5; 108.0] |
|
Note. x — p<0.05 compared to the intact group; * — p<0.05 compared to the experimental periodontitis group.
Modeling of chronic pain syndrome showed reduction of the number of adhesive macrophages in peritoneal washing cytology test by 14.9% (p<0.05) at the 3rd week of experiment compared to intact animals (Table 2). Animals with experimental periodontitis were characterized by decrease in content of macrophages in peritoneal washing cytology test by 15.7% in 7 days after removal of thread (p<0.05), but the number of adhesive macrophages was the same (p>0.05). After 14 days, both parameters decreased (content of macrophages in peritoneal washing cytology test by 42.4% (p<0.05), content of adhesive macrophages in population by 22.0% (p<0.05)) compared to intact animals. After 21 days, the content of adhesive macrophages in population did not significantly differ from this value in intact rats, but the content of macrophages was lower by 24.1% than in intact animals (p<0.05).
Table 2. Adhesive ability of macrophages in rats with experimental periodontitis and chronic pain syndrome
|
Variable Group |
Period of experiment |
Relative number of macrophages in washout, % |
Relative number of adhesive macrophages, % |
|
Intact group |
95.5 [94.5; 97.5] |
70.5 [66.5; 73.0] |
|
|
Chronic pain syndrome |
3 weeks |
93.0 [91.0; 96.0] |
60.0 [58.0; 64.0]x |
|
5 weeks |
94.5 [94.0; 96.0] |
67.5 [64.0; 73.0] |
|
|
6 weeks |
94.5 [94.0; 97.0] |
66.5 [64.0; 70.0] |
|
|
7 weeks |
93.5 [93.0; 95.0] |
66.5 [64.0; 68] |
|
|
False-operated rats |
5 weeks |
94.0 [93.0; 96.0] |
70.0 [67.0; 74.0] |
|
6 weeks |
96.5 [94.0; 98.0] |
69.0 [67.0; 73.0] |
|
|
7 weeks |
95.5 [93.0; 97.0] |
68.0 [65.0; 72.0] |
|
|
Experimental periodontitis |
7 days |
80.5 [77.5; 84.5] |
68.5 [67.5; 74.0] |
|
14 days |
55.0 [53.5; 66.0] |
55.0 [52.0; 63.0]x |
|
|
21 days |
72.5 [66.0; 75.5]x |
73.0 [69.5; 78.5] |
|
|
Experimental periodontitis in false-operated rats |
7 days |
78.0 [76.0; 83.5]x |
68.0 [64.0; 71.0] |
|
14 days |
58.5 [56.5; 64.5]x |
58.5 [56.5; 64.5]x |
|
|
21 days |
66.5 [63.5; 72.0]x |
69.5 [65.5; 73.0] |
|
|
Experimental periodontitis in chronic pain syndrome |
7 days |
65.5 [60.5; 68.0]* |
59.5 [53.5; 65.5]* |
|
14 days |
60.5 [56.0; 66.0] |
54.0 [49.5; 57.0] |
|
|
21 days |
69.0 [66.0; 72.0] |
69.5 [64.5; 72.5] |
|
Note. x — p<0.05 compared to the intact group; * — p<0.05 compared to the experimental periodontitis group.
Wistar rats with chronic pain syndrome and experimental periodontitis were characterized by significant decrease of relative number of macrophages in peritoneal washout and relative number of adhesive macrophages in population (by 18.6% (p<0.001) and 13.1% (p<0.05), respectively) after 7 days compared to the experimental periodontitis group.
Discussion
Chronic stress, including pain syndrome, is accompanied by imbalance in release of pro-inflammatory and anti-inflammatory cytokines with subsequent immunosuppression [13]. Most likely, immunosuppression realized by stress-related hormones explains decrease of functional activity of macrophages and neutrophils at the 3rd week of experimental chronic pain syndrome. In our opinion, no changes in functional activity of neutrophils and macrophages after 5–7 weeks is due to adaptive reactions blocking suppressive effect of stress-inducing hormones. Release of neuroactive peptides as antagonists of glucocorticoids has been previously reported [14]. Activation of astroglia under stress has also been established, these cells release pro-inflammatory cytokines that activate macrophages [15].
Apparently, reduction of relative number of functionally active neutrophils is associated with activation of myelopoiesis and release of a large number of young cells with reduced functional activity. At the same time, no changes of phagocytic number indicate that functionally mature cells retain high phagocytic activity. Normal phagocytic index and increase of phagocytic number compared to intact animals in 21 days after removal of thread indicate restoration of functional activity of phagocytes.
In our opinion, changes of functional activity of macrophages are due to their mobilization to the focus of inflammation and enhanced development of monocytes in bone marrow with formation of a pool of insufficiently active cells. Polarization of macrophages with predominant M2 phenotype cells under the influence of periodontopathogenic bacteria on the 14th day of experiment is also possible. These cells are characterized by weak phagocytic and functional activity compared to M1 phenotype macrophages [16]. Progressive predominance of M2 phenotype cells in long-term course of Helicobacter pylori-associated chronic gastritis has been previously reported [17]. After 21 days, proportion of M1 phenotype cells significantly increases due to less polarizing effect of microorganisms.
Decrease of functional activity of neutrophils and macrophages on the 7th day of experiment in animals with experimental periodontitis and chronic pain syndrome is apparently due to development of chronic stress in modeling of chronic pain syndrome and insufficient stress-limiting compensatory reactions. These reactions are observed in modeling of inflammatory process in periodontium. Perhaps, preliminary modeling of chronic pain syndrome contributes to predominance of M2 phenotype cells in general population of macrophages on the 7th day of experimental periodontitis.
Conclusion
We observed reduction of phagocytic activity of neutrophils and adhesive ability of macrophages at the early stages of chronic pain syndrome that is apparently associated with inhibitory effect of stress-realizing hormones. Reduced phagocytic activity of neutrophils and adhesive ability of macrophages in modeling of experimental periodontitis are associated with enhanced myelopoiesis and monocytopoiesis. These processes result a pool of young cells with reduced functional activity. Another potential aspect is predominance of M2 phenotype cells. Modeling of experimental periodontitis in animals with chronic pain syndrome is accompanied by decrease of functional activity of neutrophils and macrophages compared to the group of experimental periodontitis only in early period of experimental periodontitis. Apparently, this is due to insufficiency of stress-limiting systems or the influence of chronic pain syndrome on release of M2 pool of macrophages.
Author contribution:
Concept and design of the study — A.E. Brusentsova, Yu.D. Lyashev, N.V. Tsygan, E.B. Artyushkova
Collection and analysis of data — A.E. Brusentsova, A.Yu. Lyashev
Statistical analysis — A.E. Brusentsova, A.Yu. Lyashev
Writing the text — A.E. Brusentsova, Yu.D. Lyashev, N.V. Tsygan, A.Yu. Lyashev
Editing — E.B. Artyushkova
Funding. The study had no sponsorship.
The authors declare no conflicts of interest.