A total of 48 male Sprague-Dawley rats (9 weeks old, 300 ~ 320 g) were acquired from Beijing HFK Bioscience Co., Ltd (China) and housed in a temperature-controlled room (22 ~ 24 °C) with a 12/12 light/dark cycle and fed with standard rat chow and tap water. All procedures were approved by the Medical Ethics Committee of the Shanxi Academy of Traditional Chinese Medicine (Approval Number: SZYLY2022KY-0302). All experiments were performed in accordance with relevant guidelines and regulations, and the authors complied with the ARRIVE guidelines.

The rats were randomly divided into 6 groups, with each 8 in each group. The right hindlimb hair was removed by depilatory cream 1 day earlier. Then the rat was anesthetized with 2% isoflurane and the right knee was wrapped by a rubber tourniquet to engorge the dorsalis pedis vein for easy puncture. The chemical phlebitis was induced in rats of 5 groups by injecting 0.1 mL of 1 mg/ mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, and 5 mg/mL vinorelbine solution, respectively. While the remaining rats were received 0.1 mL of normal saline serving as sham group. The puncture site was pressed for 5 min to prevent bleeding or drug leakage after injection. Then the general symptoms of phlebitis were observed daily. On day 7, the animals were euthanized with Euthasol (100 mg/kg, IV), and a 1-cm segment of the vein extending proximally from the puncture site in the right hindlimb was collected for histopathological examination.

The volume of the injected hindlimb (2 cm above the ankle) in rat was measured by drainage method¹⁶ before modeling and post-model daily for 7 days. The edema was quantified as swelling rate, which is calculated by the following formula:

\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{Swelling~rate~(\% )~ = ~}}\frac{{{\text{Post - model~volume~}} - {\mathrm{~initial~volume}}}}{{{\mathrm{Initial~volume}}}} \times {\text{100\% }}$$\end{document}

Pain was evaluated by weight-bearing ratio of treated hindlimb, which is determined by the bipedal balance test¹⁷ before modeling and post-model daily for 7 days. Briefly, the rats were placed in the bipedal balance analgesiometer and make sure both of the hind paws stand on the corresponding pressure sensors for recording the bilateral paw pressures, respectively. The weight-bearing ratio is calculated by the following formula:

\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{Weight - bearing~ratio~(\% ) = ~}}\frac{{{\mathrm{Pressure~on~the~injected~paw}}}}{{{\mathrm{Total~pressure~of~both~paws}}}} \times {\text{100\% }}$$\end{document}

The symptoms of chemical phlebitis including erythema, pain, edema, palpable cord, and purulent drainage in the injected hindlimb were observed each day for 7 and the severity was graded according to the phlebitis scale¹⁸ shown in Table 1.

Table 1

Phlebitis grading scale.

The venous tissues were fixed in 4% polyformaldehyde, dehydrated and embedded in paraffin blocks. Subsequently, transverse sections of 5 μm were stained with hematoxylin-eosin (HE) and masson’s trichrome staining, respectively. Images were visualized using an M8 digital scanning microscopy imaging system (Lecia).

The venous tissues were pre-fixed with 3% glutaraldehyde at 4 °C for 2 days and post-fixed with 1% osmic acid for 2 h. Subsequently, the tissues were gradually dehydrated with 50–100% ethanol. For scanning electron microscopy (SEM), the longitudinal section of veins was treated by isoamyl acetate for 15 min and dried by a critical point dryer. Specimens were attached to metallic stubs by carbon stickers and sputter-coated with gold for 30 s. Then the sections were observed and captured by SEM (HITACH, SU8100). For transmission electron microscopy (TEM), transverse sections of veins were immered in 100% acetone/Epon 812 and ultrathin slices (60 nm) were prepared. The sections were stained with 5% uranium acetate for 30–60 min and then with lead citrate for 10 min. After drying, the sections were observed and captured by TEM (HITACH, HT7800).

The data were analyzed using the SPSS software version 22.0. The swelling rate and weight-bearing ratio were presented as mean ± standard deviation. Comparisons between the two groups were conducted using the independent t-test. The Kruskal-Wallis one-way analysis of variance was used to analyze the grading of phlebitis. A p-value of less than 0.05 was considered statistically significant.

The general symptoms in hindlimb of rats following intravenous injection of saline or vinorelbine via the dorsal pedal vein were observed daily for 7 days. Representative photographs were captured every other day, as depicted in Fig. 1. During observation period, sham-operated rats (0 mg/mL) exhibited no signs of erythema or edema in the hindlimb with skin intact. In contrast, rats administered vinorelbine displayed marked erythema and edema as early as day 1 post-injection, with symptom severity escalating in a dose-dependent manner. By day 3, these manifestations progressed, accompanied by ulcer formation. Subsequently, partial alleviation of erythema and edema was observed by day 5. However, these improvements were followed by the development of induration and streak formation by day 7.

Fig. 1

The general symptoms of injected hindlimb in rats induced by different concentrations of vinorelbine via dorsal pedal vein, with images taken every other day.

As illustrated in Table 2, the swelling rate of hindlimb in rats treated by all concentration of vinorelbine significantly increased compared to the sham rats on the 1st day post-injection. Furthermore, the swelling rate increased in accordance with the ascending concentration of vinorelbine solution and the peak was observed on the 3rd day. After that, the swelling rate exhibited a slight decrease on the 5th day but still remained above 30% in the 3 mg/mL, 4 mg/mL and 5 mg/mL vinorelbine groups on the 7th day.

Table 2

Comparison of swelling rates induced by different concentration of vinorelbine.

Pain was identified when the weight-bearing ratio of injected paw in bipedal stance testing fell below 50%. As shown in Table 3, the weight-bearing ratio of injected paw in sham group maintained about 50% during the experimental period. In contrast, the ratio in rats injected with vinorelbine solution may less than 50%, the extent of which depended on the timepoint and vinorelbine concentration. On the 1st day, the ratio in rats of all vinorelbine-treated groups with a slight decrease tendecy was observed compared to the sham rats. Obviously, the ratio was decreased markly on the 3rd and 5th day, especially in the 3 mg/mL, 4 mg/mL and 5 mg/mL vinorelbine groups. By the end of day 7, the ratio of injected paw in rats treated by 4 mg/mL and 5 mg/mL vinorelbine still significantly decreased compared to the sham rats.

Table 3

The weight-bearing ratio of the injected hindlimb in rats.

The chemical phlebitis in each rat was graded by phlebitis scale on day 7 and the results were shown in Table 4. The severity of chemical phlebitis among these groups was correlated with the concentration of vinorelbine solution. All the rats in sham group (0 mg/mL) and 1 mg/mL vinorelbine group were graded 0 with no symptoms. While in the 2 mg/mL vinorelbine group, 37.5% of the rats were graded 0 and 62.5% displayed grade I. The incidence of grade I, II and III in the 3 mg/mL group were 25%, 50% and 25%, respectively. In the 4 mg/mL group, the incidence of grade II, III, and IV were 25%, 50% and 25%, respectively. The grade III and IV account for 37.5% and 62.5% in the 5 mg/mL group, respectively.

Table 4

The grading of chemical phlebitis assessed by phlebitis scale on day 7.

Hematoxylin-eosin staining was performed on venous tissues sampled from the injected hindlimb of rats on day 7 with representative graphs shown in Fig. 2. Venous tissues from 0 mg/mL exhibited normal morphology, characterized by flexible vessel walls with classical layers of tunica adventitia, media and intima. The intima displayed hobnailed endothelium and the lumen was free of thrombus. Veins from rats treated with 1 mg/mL vinorelbine showed nearly identical histological features to normal veins, with no significant pathological alterations. However, the thicken vein wall were presented in the venous tissues collected from the rats injected 2 mg/mL vinorelbine. More pronounced pathological changes were observed in the 3, 4, and 5 mg/mL vinorelbine groups. These veins exhibited marked wall thickening, luminal stenosis, and thrombi within the circular venous lumen, indicating progressive dose-dependent venous damage.

Fig. 2

Representative histopathology images of venous tissues collected from rat hindlimbs stained by hematoxylin-eosin. above, 80×magnification, scale bar = 100 μm; below, 400×magnification, scale bar = 20 μm.

On day 7 post-injection, longitudinal venous tissues from rat hindlimbs were examined by scanning electron microscopy as shown in Fig. 4. In both 0 mg/mL and 1 mg/mL vinorelbine-treated rats, venous tunica intima exhibited a smooth, continuous surface composed of polygonal endothelial cells aligned parallel to the vascular longitudinal axis. Notably, local tunica intima in the 2 mg/mL vinorelbine group adhered to a substantial number of activated platelets. At higher concentrations (3–5 mg/mL), more pronounced dose-dependent mirstructural alterations of lumen surface was observed, characterized by rough with blood cells, densely packed spindle-shaped endothelial cells, and significant accumulation of extracellular matrix.

Fig. 4

Representative microstructure images of vascular intima observed by scanning electron microscopy. above, 500×magnification, scale bar = 100 μm; below, 3000×magnification, scale bar = 10 μm.