Changes in the indicators of connective tissue metabolism in the blood serum of experimental rats under the conditions of modeling the development of degenerative processes in paravertebral muscles

Low back pain is a common health problem. To deepen the understanding of the pathogenesis of the disease, experimental studies on animals with modeling of the pathological process are necessary. Objective. Based on the analysis of biochemical markers of connective tissue metabolism in the blood serum of laboratory rats, the applicability of the studied models of degenerative muscle tissue damage to study the relationship between this condition and the development of disorders in spinal motor segments was assessed. Methods. Two models of reproduction of degenerative processes in the paravertebral muscles of white rats were tested: I (n = 5) — alimentary (diet-induced) obesity, by keeping it for 3 months on a high-calorie diet; II (n = 5) — ischemia, by tying the large rectus muscles of the back with suture material (45 days). Control group (n = 5) — intact animals of similar age and sex. The content of glycoproteins, total chondroitin sulfates (CS), hexosamines, protein-bound hexoses, seroglycoides, fractional distribution and total content of hydroxyproline and glycosaminoglycan


Introduction
Lumbar pain is a common health problem, affecting more than 80 % of adults during their lifetime and is the leading cause of disability worldwide. This condition is often associated with lumbar disc degeneration, which should not be considered as an isolated event, but rather a continuum of events. Patients with significant intervertebral disc degeneration often have increased fatty infiltration in the multipartite and extensor muscles. In women, it is more severe at the level of L IV -L V and L V -S I . In men, fatty infiltration is greater in the lumbar muscle at the L V -S I level [1,2].
The muscles that attach to the spine play a crucial role in the functioning of the spine and the entire body. A clear association between fatty infiltration and paravertebral muscle fibrosis and low back pain has been demonstrated in the scientific literature. Dysfunction/degeneration of muscles can be a factor in initiating the progression of spine abnormalities, in particular, disorders in the structure of the intervertebral disc [3]. Conversely, dysfunction of the lumbar paravertebral muscles due to pain, caused by structural and functional changes in the spinal motor segments, can trigger a violation of their structure. Macroscopically, it is manifested by a decrease in the cross-sectional area and an increase in fat infiltration in the paravertebral muscles at the lumbar level [1,4]. In addition, there are microscopic changes, such as an impairment of the distribution of fibers [5].
Vertebral lesions are associated with fatty infiltration of the paravertebral muscles. It can be assessed qualitatively (e.g. Gutalier's classification) and quantitatively using software for calculating MRI scans or computed tomography [6,7].
Assessing the state of the paravertebral muscles before surgery for degenerative spine diseases can be useful for the surgeon in terms of predicting the functional state and recovery of patients [5,8].
The purpose of the study: based on the assessment of biochemical indicators of connective tissue metabolism in the blood serum of laboratory rats, to assess the suitability of the studied models of degenerative muscle tissue damage for further study of the relationship between this condition and the development of degenerative disorders in spinal motor segments.

Material and methods
Experimental studies were conducted in compliance with the requirements of humane treatment of experimental animals [9,10]  Within the framework of the study, two variants of reproduction of degenerative processes in paravertebral muscles were tested using female white laboratory rats (at the beginning of the experiment, aged 2 months, weight 130-210 g) of the population of the above experimental biological institution.
In the first group (model of alimentary (diet-induced) obesity) rats (n = 5) were kept for 3 months on a high-calorie diet adapted to the Teklad Custom Diet TD.10670 formulation (22.5 g or 40-45 % kcal fat), developed by the Envigo company for laboratory rats and mice [4,11]. Food composition per 100 g: lard 18 g, pork liver 2 g, sunflower oil 3 g, wheat groats 45 g, cottage cheese 2 g, egg powder 10 g, milk powder 6 g, sugar 5 g, beetroot 3 g, carrot 2 g, meat and bone meal 4 g.
In the second group (ischemia model), rats (n = 5) had the large rectus muscles of the back tied with suture material through a skin incision (Fig. 1). The animals were removed from the experiment 45 days after manipulation.
The control group (n = 5) involved intact animals of similar age and sex, which were kept on a standard diet [12].
Euthanasia of all rats was performed by decapitation under open inhalation anesthesia with diethyl ether due to the need to obtain blood for biochemical studies, which, after natural coagulation, was freed from the formed elements by centrifugation for 15 min at 3000 rpm. The supernatant liquid was separated and the content of glycoproteins was determined according to the method of Steinberg and Dotsenko [13], total chondroitin sulfates according to the reaction with rivanol using the Nemeth-Csoka method as modified by L. I. Slutsky [13,14].
Fractional analysis and research of the total content of hydroxyproline in the blood serum of experimental Fig. 1. Appearance of the surgical wound after ligation of the rectus major muscles of the back in white rats animals with the determination of fractions of free and protein-bound metabolites, which characterized anabolic and catabolic processes in the collagen-hydroxyproline system, was carried out with separation into free and protein-bound fractions [15].
The content of hexosamines in the cartilage tissue of experimental animals was determined by the method of N. F. Boas, the content of hexoses bound to protein by the orcinol method [16].
The fractional composition of glycosaminoglycan sulfates (GAGs) was determined by precipitation with resoquin and sequential separation from the sediment with sodium chloride solutions of increasing concentration. The composition of fraction I included easily soluble GAGs with a predominance of hyaluronates and chondroitin-6-sulfate, fraction II comprised moderately soluble GAGs with a predominance of chondroitin-4-sulfate, fraction III involved mainly highly sulfated glycosaminoglycan sulfates with a predominance of keratan sulfates, as well as dermatan sulfate, heparan sulfate, etc. [17]. The content of sialic acids [16], seroglycoides [18] in blood serum was also determined.
The results of biochemical studies were statistically processed according to the non-parametric Mann-Whitney test. After that, the level of error of the first kind was determined for comparing the indicators of the four groups among themselves (Kruskal-Wallis criterion), r c -the level of the error of the first kind for comparing the indicators     of the group with the control, r 1 -for comparing the indicators of the group with mechanical ischemia of muscles with non-resident suture material, r 2indicators of the group with mechanical ischemia of muscles with resident suture material (Mann-Whitney criterion). The difference was considered significant at p < 0.05 [19].

Results and their discussion
In both studied models of paravertebral muscle degeneration, no significant changes were observed in such indicators of connective tissue metabolism as the level of protein-bound hydroxyproline, proteinbound hexoses, hexosamines, total chondroitin sulfates, sialic acids, and seroglycoids (see Table).
The only indicator that showed a statistically significant difference between the obesity model and the control group was the serum level of glycoproteins. It turned out to be higher in rats kept on a hyperlipidemic diet, which, in our opinion, indicated to a greater extent the elements of intoxication in the considered conditions. The level of glycoproteins was even higher in the blood serum of rats of the second group (ischemia model) (Table, Fig. 2). In conditions of unchanged other indicators of the inflammatory process, it should probably be considered as