In vitro and in vivo neurotoxic effects of paclitaxel

Paclitaxel (PTX) is a widely used anticancer agent. In this study in vitro and in vivo neurotoxic effects of PTX were investigated. To investigate the in vitro neurotoxic effects; normal human astrocyte cells were used. After culturing astrocytes, with increasing doses of PTX for 24 and 48 hours, IC50 values were determined as 31.68 µg/ml, and 15.71 µg/ml respectively. For in vivo neurotoxic effects 27 male adult BALB/c mice were used. Animals were weighted each day and divided into three groups randomly: control (Saline, n=11), paclitaxel (PTX, n=8), vehicle (Vehicle, n=8). To PTX group, on 0th, 4th, 8th and 9th days, total of four intraperitoneal (i.p.) injections were given. Saline and Vehicle groups received same volume of serum physiologic or vehicle as i.p. Behavioral tests; hot plate and tail flick were applied to animals on one day prior to injections, 0th, 1st, 4th, 5th, 8th and 9th days. On last day of experiments, mice were sacrificed under anesthesia by cervical dislocation. Sciatic nerve and brain cortex tissues were taken. Right sciatic nerves and brain cortex hemispheres were used for histology/immunohistology. These tissues used for TUNEL apoptosis assay and, NFκB, iNOS, VEGF protein expressions on histologic level. Left sciatic nerves and brain hemispheres were used in qRT-PCR and microarray experiments. Caspase-3, -8 and -9 mRNA and total mRNA expression levels were investigated with aforementioned molecular experiments. During experiments PTX administration didn't affected the body weights of animals significantly when compared to others. According to the behavior experiment results; for hot plate results, PTX group had significant shorter response times on 8th day compared to Vehicle, on 9th day compared to both Saline and Vehicle groups. PTX created sensitivity on animals' peripheral nervous system. According to tail flick results there is no significant difference between groups. According to the qRT-PCR results; on sciatic nerves caspase-3 and caspase-9 expressions were increased significantly in PTX and Vehicle groups when compared to Saline. For caspase-8 expressions, there is no significant difference between groups. In brain cortex tissue, there is no statistical difference between all groups according to their caspase-3, -8 and -9 expression levels. These results showed PTX and vehicle activated the intrinsic apoptotic pathways in sciatic nerve. However there is no apoptotic effect of PTX and vehicle at the mRNA level in brain cortex. According to microrray results; besides an intrinsic pathway element cytochrome-c was determined as significantly expressed in sciatic nerve tissues of PTX and Vehicle groups when compared to Saline. According to Hematoxylene&Eosine (H&E) staining; for sciatic nerve there is repletion in vessels of PTX group epineurium, thickness due to fibrosis in perineurium, mild edema in endoneurium were observed. There is no histologic difference in sciatic nerves of other groups with H&E. For brain cortices there is no histological difference between all groups for H&E staining. According to TUNEL results in sciatic nerves apoptotic isles increased dramatically with PTX, also there is increase in apoptotic isles with Vehicle. According to iNOS staining results, there is no difference between groups for brain tissues; however for sciatic nerve PTX application increased the iNOS expression when compared to other groups. There is no significant difference between groups in brain and sciatic nerve tissues according to their NFκB staining results. Both in brain and sciatic nerves, VEGF expression increased in PTX and Vehicle groups when compared to Saline, this increase is slightly more in PTX. It is possible to speculate that partially vehicle and dominantly PTX has damaging effects on healthy brain and sciatic nerve tissues. We demonstrated the damaging effects of PTX application on healthy sciatic nerve at behavioral, biochemical and molecular level.