Fig 1.
In vitro neutralization assay using rabbit anti-hyaluronidase serum.
Hyaluronidase activity (%) was measured using a turbidimetric assay. Commercial hyaluronidase from bovine testis was used as a positive control, and ultrapure water was used as a negative control. Enzymatic activities of TsV (2 μg) and native hyaluronidase from TsV (0.5 μg) were tested. For the in vitro neutralization assay, TsV (2 μg) was incubated with pre-immune serum (10 μl) or anti-hyaluronidase serum (Anti-Hyal, 10 μl) for 1 h at 37°C before testing. Anti-hyaluronidase serum neutralized the hyaluronidase activity in TsV. All values are expressed as the mean ± S.E.M. of duplicates from three independent experiments.
Fig 2.
In vitro stability of 99mTc-TsV.
Stability of the complex 99mTc-TsV over time in the presence of saline 0.9% (w/v) at room temperature and in the presence of plasma at 37°C. All values are presented as the mean ± S.E.M. of duplicates from three independent experiments.
Fig 3.
Blood clearance of 99mTc-TsV. 3.7 MBq of 99mTc-TsV diluted in PBS (99mTc-TsV + PBS) or neutralized with anti-hyaluronidase serum (99mTc-TsV + Anti-Hyal serum) was intramuscularly injected in Swiss mice (6–8 weeks old, 24–28 g; n = 6 per group).
Radioactivity levels were measured in blood samples at 1, 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, and 1440 min post-injection. Data are represented as the mean percentage of the injected dose of 99mTc-TsV per gram of blood (% ID/g) ± S.E.M. of the mean. Values represent duplicates from two independent experiments. Statistical analyses were performed using a linear mixed model. Serum administration (p < 0.0001), time (p < 0.0001), and their interaction (p < 0.0001) had a statistically relevant effect on the mean 99mTc-TsV blood clearance.
Fig 4.
99mTc-TsV spreading in mice over time.
Representative scintigraphic images of mice injected with 18 MBq 99mTc-TsV diluted in PBS (A) or neutralized with anti-hyaluronidase serum (B). Samples were intravenously injected in Swiss mice (6–8 weeks old, 24–28 g; n = 3 per group). Radioactivity levels were measured 30, 60, and 120 min post-injection. Images show a quick and growing spread of 99mTc-TsV diluted in PBS over time (A). On the other hand, TsV neutralized with anti-hyaluronidase serum remains at the injection site (right tight muscle) (B). Images are pseudocolored according to the color scale.
Fig 5.
99mTc-TsV (3.7 MBq) diluted in PBS (99mTc-TsV + PBS) or neutralized with anti-hyaluronidase serum (99mTc-TsV + Anti-Hyal serum) was intramuscularly injected in Swiss mice (6–8 weeks old, 24–28 g; n = 6 per group). Radioactivity levels were measured in the heart, liver, lungs, spleen, thyroid and kidneys at 30, 60, 240 and 1440 min post-injection. The results are expressed as the percentage of injected dose/g of tissue (%ID/g). All values are presented as the mean ± S.E.M. of two independent experiments. Statistical analysis was performed using two-way ANOVA (factors: serum administration and time). Anti-hyaluronidase serum significantly affected the mean distribution of TsV to the liver (p < 0.0001), spleen (p = 0.0115), and kidneys (p = 0.0009), while time was a significant factor for TsV distribution to the heart (p < 0.0001), liver (p < 0.0001), lungs (p = 0.0095), spleen (p = 0.0008), and kidneys (p < 0.0001). A significant interaction between serum administration and time was observed in the heart (p = 0.00003), liver (p < 0.0001), spleen (p = 0.0337), and thyroid (p < 0.0001).
Fig 6.
Hyaluronidase neutralization as a first-aid treatment for scorpion sting.
3.7 MBq of 99mTc-TsV diluted in PBS was intramuscularly injected in Swiss mice (6–8 weeks old, 24–28 g; n = 6 per group). Subsequently, anti-hyaluronidase serum was injected in the same site of 99mTc-TsV injection at different times (Anti-Hyal serum; 0, 10, and 30 min post-injection; arrows indicate the injection times). Radioactivity levels in the bloodstream were measured at 1, 5, 10, 15, 20, 30, 45, 60, 90, 120, and 240 min post-injection of 99mTc-TsV. All data are expressed as the mean percentage of the injected dose of 99mTc-TsV per gram of blood (% ID/g) ± S.E.M. of the mean. Values are representative of duplicates from two independent experiments. Statistical analysis was performed using a linear mixed model. Time (p < 0.0001) and time x Anti-Hyal serum administration interaction (p < 0.0001) had a statistically relevant effect on the mean 99mTc-TsV blood clearance.