Method for modeling limb lymphedema in laboratory animals

Abstract

FIELD: experimental lymphology.SUBSTANCE: in Wistar rats, 0.3 ml of a 3% lauromacrogol 400 solution is injected into the popliteal and inguinal lymph nodes of one limb through skin incisions in the popliteal and inguinal regions without performing lymph node dissection.EFFECT: development of a model of pelvic limb lymphedema in experimental animals, providing a reproducible and morphologically reliable picture of chronic lymphatic insufficiency without the use of surgical removal of lymphatic structures or ionizing radiation.1 cl, 6 dwg, 1 ex

Description

[0001] This invention relates to medicine, specifically experimental lymphology, and can be used as a model of chronic lymphatic insufficiency to study the pathogenesis, develop new treatments, and effectively prevent lymphedema. Experimental lymphedema models in laboratory animals play a key role in studying the pathogenesis of the disease. However, the scientific literature is acutely short of reproducible and clinically relevant lymphedema models, particularly those reflecting the morphofunctional changes characteristic of the chronic stage.

[0002] Currently, various methods for modeling lymphedema in laboratory animals exist; however, they all have significant limitations. The earliest models were based on mechanical removal of regional lymph nodes and damage to the lymphatic vessels (Rysznyak J., 1967). This approach ensures blockade of lymphatic drainage, but is associated with high trauma, significant variability in the extent of intervention, the risk of postoperative complications, and mortality. To overcome these shortcomings, a combined radiation-surgical model was proposed: first, the lymph nodes are removed, followed by local X-ray irradiation of the limb (Lee-Donaldson L., 1999). Irradiation suppresses the formation of collateral lymph drainage, but requires specialized equipment and causes nonspecific tissue damage. An alternative option is preliminary X-ray irradiation followed by lymphadenectomy (Kanter M., 1990).This model also retains all the disadvantages associated with ionizing radiation and increases the duration of the experiment.

[0003] Thus, all known prototypes are characterized by:

[0004] - high trauma and / or toxicity;

[0005] - the need for expensive equipment;

[0006] - insufficient reproducibility;

[0007] - lack of intra-individual control.

[0008] We have proposed a method for modeling limb lymphedema in experimental animals.

[0009] The technical result of the present invention is the development of a method for modeling pelvic limb lymphedema on experimental animals, providing a reproducible and morphologically reliable picture of chronic lymphatic insufficiency without the use of surgical removal of lymphatic structures or ionizing radiation.

[0010] Implementation of the invention

[0011] The experiment was conducted on male Wistar rats, divided into an experimental group (n=7) and a control group (n=7). Control group animals were not subjected to any surgical or pharmacological treatment and were maintained under standard conditions. After anesthesia, the following manipulations were performed on the left pelvic limb of the experimental group animals:

[0012] A skin incision up to 1 cm long is made in the popliteal region, the regional popliteal lymph node is isolated and 0.3 ml of a 3% lauromacrogol 400 solution is injected into it using an insulin syringe (30G needle).

[0013] In the groin area of ​​the same limb, a skin incision up to 3 cm long is made, the inguinal lymph nodes and subcutaneous fat are isolated and 0.3 ml of a 3% lauromacrogol 400 solution is injected.

[0014] Lymph node dissection is not performed; all lymph nodes are left in situ. The wounds are sutured. The animals are returned to the vivarium and provided with postoperative care.

[0015] Thus, in animals of the experimental group:

[0016] • The left pelvic limb serves as a model of lymphedema;

[0017] • The right pelvic limb acts as an intact limb (within the individual control).

[0018] The model was evaluated after 60 days, since it is by this time that the formation of the chronic stage of lymphedema is completed, which is confirmed histologically (interstitial edema, fibrosis, macrophage infiltration, epidermal hyperplasia, proliferation of adipose tissue).

[0019] After 60 days, anthropometric monitoring is performed at 7 points (AG): the circumferences of the left (operated) and right (intact) pelvic limbs are measured at seven standard points (AG: 1-4 cm above the calcaneus in 0.5 cm increments) (Fig. 1). The data are also compared with the values ​​in animals in the control group, for which both limbs are measured.

[0020] Statistical analysis (paired t-test) showed a significant increase in circumference in the left limb compared to the right:

[0021] - A: +0.59 cm (p=0.0003, +23.2%);

[0022] - B: +0.56 cm (p=0.0012, +12.4%);

[0023] - C: +0.14 cm (p=0.0004, +2.4%);

[0024] - D: +0.13 cm (p=0.035, +2.0%);

[0025] - E: +0.29 cm (p=0.008, +4.4%);

[0026] - F: +0.24 cm (p<0.0001, +3.6%);

[0027] - G: +0.44 cm (p=0.0002, +6.3%).

[0028] The total volume (sum of circumferences) increased by an average of 2.4±0.7 cm (p=0.000074, +6.0±1.7%). In the control group, no differences were found between the limbs (p>0.05).

[0029] At the end of the 60-day period, animals are removed from the experiment. Skin, subcutaneous tissue, and underlying muscle samples are collected from all animals (experimental and control groups):

[0030] in the control group - from the left and right pelvic limbs;

[0031] in the experimental group - from the left (with lymphedema) and right (intact) limbs.

[0032] Biopsy specimens are fixed in 10% neutral-buffered formalin and subjected to standard histological processing. Histological sections are stained with hematoxylin and eosin and using the van Gieson method.

[0033] Brief description of drawings

[0034] Fig. 1 shows the points for measuring the circumference of the hind limbs

[0035] Fig. 2 shows the morphological picture of the skin of rats in the control group. Hematoxylin and eosin, magnification ×100

[0036] Fig. 3 shows the morphological picture of the skin of rats of the control group. Van Gieson circle, magnification ×100

[0037] Fig. 4 shows the morphological picture of the skin of a limb with lymphedema. Hematoxylin and eosin, magnification ×100

[0038] Figure 5 shows the morphological picture of the skin of a limb with lymphedema. Hematoxylin and eosin, magnification ×400

[0039] Fig. 6 shows the morphological picture of the skin of a limb with lymphedema. Van Gieson circle, magnification ×400

[0040] Clinical Example No.1:

[0041] Histological results of the thigh skin of the rat from the experimental group, confirmed by micrographs:

[0042] On the 60th day of the experiment, the following changes were detected in the left limb of the animals in the experimental group (limb with lymphedema):

[0043] Epidermis: thickening, hyperkeratosis, mitotic figures in the basal layer, perinuclear edema in all layers, increased number of cell rows in the spinous (3-4) and granular (2-3) layers (Fig. 5).

[0044] Dermis: significant expansion of the interfibrillar space due to interstitial edema (Fig. 4, 6); collagen fibers are swollen, with signs of fraying (Fig. 6); pronounced lymphocyte-macrophage infiltration; blood filling of vessels (Fig. 4, 5).

[0045] Hypodermis: proliferation of loose fibrous connective tissue, increase in volume and number of fat cells.

[0046] Muscle tissue: thickening of connective tissue layers, more pronounced around the vascular-nerve bundles.

[0047] Clinical Example No.2:

[0048] Histological results of the thigh skin of the rat from the control group, confirmed by micrographs:

[0049] epidermis - thin, with clear layering and absence of mitotic figures;

[0050] dermis - dense, unformed connective tissue containing multidirectional bundles of collagen fibers without signs of fraying and swelling (Fig. 2, 3);

[0051] single lymphocytes, no significant edema (Fig. 2).

[0052] In the right (intact) limb of animals in the experimental group, the skin structure is virtually identical to that of animals in the control group, which did not undergo the intervention. Thus, the intact limb of animals in the experimental group exhibits a morphological pattern identical to that of the control group, confirming the localized nature of the effect of Lauromacrogol 400 and the absence of systemic effects.

[0053] The morphological picture in the left limb of the experimental group corresponds to chronic lymphedema with signs of persistent edema, fibrosis, chronic inflammation and tissue remodeling.

[0054] Advantages of the proposed method:

[0055] 1. No lymph node dissection or ionizing radiation;

[0056] 2. High reproducibility due to standardized introduction of lauromacrogol 400;

[0057] 3. Availability of intra-individual control (intact limb);

[0058] 4. Technological simplicity - does not require special equipment.

[0059] The obtained data confirm that the proposed method allows for the reproducible creation of an experimental model of chronic pelvic limb lymphedema without the use of ionizing radiation or lymph node dissection, suitable for studying pathogenesis and testing preventive and therapeutic strategies.

[0060] Bibliography:

[0061] 1. Rysznyak J., Foldi M., Szabo G. Lymphatics and Lymph Circulation. 2nd ed. Oxford: Pergamon Press, 1967. 704 p.

[0062] 2. LEE-Donaldson L., Witte MH, Bernas M., Witte CL, Way D., Stea B. Refinement of a Rodent Model of Peripheral Lymphedema / / Lymphology. 1999. Vol. 32, No. 3. P. 111-117.

[0063] 3. KANTER M., SLAVIN S., KAPLAN W. An experimental model for chronic lymphedema / / Plastic and Reconstructive Surgery. 1990. Vol. 85. P. 573-580.

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