Dynamic behavior of nerve growth factor upon administration to the rat brain parenchyma

By understanding how neurotrophins navigate through brain tissue, we move closer to unlocking the potential therapeutic benefit of these small (26-28kD), basic proteins. We utilize two-photon laser scanning microscopy (TPLSM) to characterize the temporal and spatial fate of the prototypical neurotrophin, nerve growth factor, in brain tissue. We administer a rhodamine-nerve growth factor conjugate (RNGF) from a micropipette to the striatal region of rat brain tissue slices. Two-photon imaging allows us to image deep into 400 micron-thick tissue slices with minimal photodamage of tissue and photobleaching of label. We use a point-source diffusion model to describe the distribution of RNGF and estimate an apparent brain tissue diffusion coefficient (D_B(34°C)) of 2.75×10^-7cm²/sec. We utilize two-photon fluorescence photobleaching recovery (FPR) to estimate the free diffusion coefficient in water (D_F(34°C)) of 8.79×10^-7cm²/sec. The extracellular space creates a tortuous path for diffusion, and an anticipated mismatch between D_F and D_B. The square-root of the ratio of D_F to D_B gives the tortuosity, τ. We compare our estimate of τ (1.79) to literature values for other proteins, and conclude that NGF enjoys relatively rapid diffusion in tissue. We extrapolate these findings to postulate formulation strategies for eventual clinical application.