Multiple sclerosis (MP) is a sever neurodegenerative autoimmune disorder. Due to its high prevalence and the severity of symptoms causing partial or complete loss of mobility, multiple sclerosis remains a pressing problem, prompting a search for new therapies. Most patients with MS completely loose the mobility 25 years after the onset of the disease. More than a half of MS patients become dependent on crutches 15 years after appearance of the first symptoms. To date, there is no effective causal treatment for MS.

Usually the disease strikes at young age: 70 % to 80 % of patients suffer the first symptoms of MS between 20 and 40 years of age [1]. MS is diagnosed by neurological examinations, magnetic resonance imaging of the central nervous system, and by biopsy or autopsy [2]. MS has numerous clinical manifestations indicating damage to the spinal cord, the brain, cranial nerves, the cerebellum, and cognitive function. Current diagnostics are insufficient for accurate estimation of MS severity. MRI, electroencephalography and lumbar puncture can still be inconclusive, in spite of providing valuable information about patient’s condition. In patients with MS, many symptoms can be caused by infection, vascular pathology, or autoimmune comorbidities [3].

There are four types of MS: relapsing-remitting (RRMS, alternating periods of relapses and remissions) occurring in 80 % to 85 % of patients; primary progressive (PPMS) occurring in 10 % to 15 % of patients; progressive-relapsing (PRMS) — in 5 % of patients; and secondary-progressive (SPMS) [4, 5]. About half of patients with RRMS develop symptoms of SPMS 10 years after the onset of the disease. Over 90 % of patients with RPMS eventually demonstrate SPMS symptoms [6].

The hallmark of MS is destruction of the myelin sheaths of neurons in the central nervous system caused by clustering T- and B-cells. Another typical feature of this disease is accumulation of oligoclonal antibodies in the cerebrospinal fluid. It is not clear, though, how and where the clonal expansion of lymphocytes specific for myelin basic protein is initially triggered. We do not know yet whether it happens in the CNS, where the myelin sheath is directly involved, or outside of it, with autoreactive species migrating to the CNS from other places [7].

Development of effective MS treatments is impossible without animal models accurately replicating the course of the disease in humans, such as experimental autoimmune encephalomyelitis (EAE) of rats and mice. EAE is induced by injecting myelin or basic myelin protein (MBP) suspensions in incomplete Freund’s adjuvant into the hind footpads of rodents [8]. One month after immunization the mice develop hind limb paralysis which lasts for 4–6 months [9]. In Dark Agouti (DA) rats, EAE progresses more rapidly (paralysis sets in on days 10–11 and lasts until day 14). The key difference of EAE in animals from MS in humans is full recovery of rodents, which is absolutely unattainable for humans at this point.

An interesting study [10] reports cytokine profiles of 19 patients with MS, including 16 patients with RRMS, 1 individual with PPMS, and 2 — with SPMS. The patients were distributed into groups based on disease duration from the moment of diagnosis: 4.2 ± 0.8 months in group 1 and 76.6 ± 14.3 months in group 2. The study showed that in earlier stages of MS (in comparison with later stages and the absence of the disease), interferon gamma (IFNγ) and the anti-inflammatory lymphokine IL-10 dominate in the cytokine profiles. In the late stage, the levels of IL-1RA, IL-8, IL-12(p70), CCL-3, CCL-7, CCL-11, CXCL-10, FGF, and IFNγ go down. Later stages are also characterized by elevated levels of IL-1a, IL-1b, IL-2RA, IL-3, IL-4, IL-7, IL-12(p40), IL-18, CCL-5 (RANTES), CCL-27, HGF, MIF, M-CSF and TRAIL. Interestingly, MS patients were shown to have elevated blood levels of IL-17, known to play a key role in triggering development of psoriatic skin lesions [11]. In addition, patients with RRMS exhibited elevated IL-22 levels. Dynamics of cytokine profiles in the cerebrospinal fluid drove the researchers [10] to the conclusion about the crucial role of the accumulating IFNγ and MIF (a key factor of joint capsule degeneration in osteoarthritis) and a few other factors stimulating migration of lymphocytes: CCL-5 (RANTES), CCL-2 and CCL-27, induced by IFNγ and MIF. The study also revealed accumulation of proapoptotic TNFα and TRAIL- ligand in the cerebrospinal fluid (but not blood) of MS-stricken patients.

These data suggest a few patterns typical for MS, including increased long-term systemic activity of hematopoietic growth factors, in particular those targeting granulocytes, sustained Th1-response, and overrepresentation of lymphocyte/ monocyte migration factors in the absence of pronounced proinflammatory response (factors stimulating production and taxis of neutrophils). The study [10] could provide an insight into how cytokine levels observed in the cerebrospinal fluid and blood change in patients with MS, but due to the limitations of the applied statistical methods, significance of the identified patterns is questionable.

Considering the above said, our study aimed to

1. investigate the short-term dynamics of cytokines in rats with rapidly progressing induced EAE;
2. compare the data on cytokine levels in patients with MS and in rats with induced EAE in order to assess the feasibility of the EAE rat model for testing anti-MS candidate drugs.

METHODS

Induction of EAE in rats

Experiments involving laboratory animals were carried out in compliance with the “Regulations for the use of Experimental Animals” (Addendum to Order 755 of the Ministry of Health of the USSR dated August 12, 1977) and the principles of the Declaration of Helsinki (2013).

Homogenates of the spinal cord of random-bred rats were prepared as described in [12]. Further in vivo experiments were carried out in Dark Agouti rats weighing 220–250 g. The main group included 11 animals. On day 0 the animals were injected with the spinal cord homogenate mixed with incomplete Freund’s adjuvant in the ratio of 1 : 1 into the hind footpads. The total volume of the injected mixture was 100 μl per paw. The controls (n = 7) received 100 μl of normal saline mixed with incomplete Freund’s adjuvant in the ratio of 1 : 1. From day 1 through day 7, except for day 6, blood samples were collected from the tail vein (500 μl of blood daily) and immediately used for serum preparation. Briefly, blood was placed into Vacuette Z serum sepclot activator vacuum test tubes and centrifuged for 15–20 min at 2,500 rpm and +4 °С. The obtained serum (about 100 μl) was transferred to microcentrifuge tubes and frozen at –20 °С. The animals were weighted daily, and the severity of the disease was assessed using the following scale: 0 points — no symptoms, 1 point — decreased tail tone, 2 points — impaired righting reflex, 3 points — partial paralysis, 4 points — complete paralysis, 5 points— moribund or dead. In borderline cases, a lower index value was opted. Clear signs of EAE appeared in the controls starting from day 8 to day 14 of the experiment. On days 11–14 the disease reached its peak, which lasted for 2–3 days.