Pharmacokinetics and bioactivity of glial cell line-derived factor (GDNF) and neurturin (NTN) infused into the rat brain

Abstract

Convection-enhanced delivery (CED) of GDNF and NTN was employed to determine the tissue clearance of these factors from the rat striatum and the response of the dopaminergic system to a single infusion. Two doses of GDNF (15 and 3 microg) and NTN (10 microg and 2 microg) were infused into the rat striatum. Animals were euthanized 3, 7, 14, 21, and 28 days post-infusion. Brains were processed for ELISA, HPLC, and immunohistochemistry (IHC). Both doses of the infused GDNF resulted in a sharp increase in striatal GDNF levels followed by a rapid decrease between day 3 and 7. Interestingly, IHC revealed GDNF in the septum and the base of the brain 14 days after GDNF administration. Dopamine (DA) turnover was significantly increased in a dose-dependent manner for more than 7 days after a single GDNF infusion. NTN persisted in the brain for at least two weeks longer than GDNF. It also had more persistent effects on DA turnover, probably due to its precipitation in the brain at neutral pH after infusion. Our data suggest that daily or continuous dosing may not be necessary for delivering growth factors into the CNS.

Fig. 1

Pharmacokinetics of GDNF and NTN infused into the rat brain – study design. The scheme shows our experimental design for the striatal infusion of GDNF and NTN proteins. Two different doses of GDNF and NTN were used (15 μg and 3 μg for GDNF; 10 μg and 2 μg for NTN). Rats were euthanized at different time points to measure the tissue levels of GDNF and dopamine turnover as a response to a single GDNF and NTN infusion. The brains were processed for ELISA (only for the rats infused with GDNF), HPLC, and IHC staining (see details in Material and Methods).

Fig. 2

The tissue clearance of GDNF protein from the rat brain after a single GDNF infusion. The upper panel with darker columns represents the high dose group (rats were infused with 15 μg GDNF). The lower panel with lighter columns shows the results from the low dose group (rats were infused with 3 μg GDNF).

Fig. 3

Immunostaining for GDNF infused into the rat brain – time course. Columns A, B, and C show the time points after striatal infusions – 3, 7, and 14 days, respectively. GDNF was infused bilaterally – 15 μg into the left and 3 μg into the right hemisphere. No signal was detected 21 and 28 days after infusion. The arrows in column C show residual GDNF staining (septum and the base of the brain including retrochiasmatic area). The size bar = 1.5 mm.

Fig. 4

Immunostaining for GDNF infused into the rat brain. (A) Higher magnification from the frame in column A – striatal neurons positive for GDNF; size bar = 100 μm. (B) Higher magnification from the frame in column B – GDNF staining in the vicinity of a blood vessel. After striatal infusion, GDNF was transported to a different brain region (in this case - dorsal endopiriform nucleus) via perivascular transport, phenomenon we often observe during cerebral infusion of vectors and other molecules; size bar = 100 μm.

Fig. 5

Turnover of dopamine (DA) after a single infusion of GDNF protein into the rat striatum. The upper panel shows the results for the high dose group (15 μg GDNF into the left hemisphere), the lower panel represents the low dose group (3 μg GDNF into the left hemisphere). The turnover of dopamine is expressed as the ratio of dopamine metabolites, (3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) to DA itself. Darker columns represent results from the GDNF-infused hemispheres, lighter – from the control, right, hemispheres infused with PBS. Statistically significant differences are marked as * and p values are given.

Fig. 6

Immunostaining for NTN infused into the rat brain – time course. Columns A, B, C, and D show the time points after striatal infusions – 3, 7, 14, and 21 days respectively. NTN was infused bilaterally – 10 μg into the left and 2 μg into the right hemisphere. No signal was detected 28 days after infusion. The left hemisphere was infused with a higher dose (10 μg) and the right one with a lower dose (2 μg).

Fig. 7

Turnover of dopamine (DA) after a single infusion of NTN protein into the rat striatum. The upper panel shows the results for the high dose group (10 μg NTN into the left hemisphere), the lower panel represents the low dose group (2 μg NTN into the left hemisphere). The turnover of dopamine is expressed as the ratio of dopamine metabolites, (3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) to DA itself. Darker columns represent results from the NTN-infused hemispheres, lighter – from the control, right, hemispheres infused with PBS. Statistically significant differences are marked as * and p values are given.