Targeted expression of a multifunctional chimeric neurotrophin in the lesioned sciatic nerve accelerates regeneration of sensory and motor axons

Abstract

Peripheral nerve injury markedly regulates expression of neurotrophins and their receptors in the lesioned nerve. However, the role of endogenously produced neurotrophins in the process of nerve regeneration is unclear. Expression of a multifunctional neurotrophin, pan-neurotrophin-1 (PNT-1), was targeted to the peripheral nerves of transgenic mice by using a gene promoter that is specifically activated after nerve lesion but that is otherwise silent in all other tissues and during development. PNT-1 is a chimeric neurotrophin that combines the active sites of the neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 and binds and activates all known neurotrophin receptors. In adult transgenic mice, PNT-1 was highly expressed in transected but not in intact sciatic nerve. Morphometric analyses at the electron microscopy level showed increased and accelerated recovery of axon diameter of myelinated fibers in crushed peripheral nerves of transgenic mice compared with wild type. Examination of nerve bundles in target tissues indicated accelerated reinnervation of foot pad dermis and flexor plantaris muscle in transgenic mice. Moreover, transected sensory and motor axons of transgenic mice showed faster and increased return of neurophysiological responses, suggesting an accelerated rate of axonal elongation. Importantly, transgenic mice also showed a markedly ameliorated loss of skeletal muscle weight, indicating functional regeneration of motor axons. Together, these data provide evidence, at both the anatomical and functional levels, that neurotrophins endogenously produced by the lesioned nerve are capable of significantly accelerating the regeneration of both sensory and motor axons after peripheral nerve damage. In addition, our results indicate that exogenous PNT-1 administration may be an effective therapeutic treatment of peripheral nerve injuries.

Figure 1

Delivery of PNT-1 to the lesioned sciatic nerve by overexpression in transgenic mice. (A) Schematic representation of the PNT-1 expression construct. Hatched bar, upstream sequences of the fourth promoter of the BDNF gene; solid bar, coding region of the PNT-1 gene; open bar, rabbit β-globin gene intron and polyadenylation sequences. (B) Expression of PNT-1 mRNA in liver, intact sciatic nerve, and 1-week-lesioned sciatic nerve of transgenic mice analyzed by RPA.

Figure 2

Morphometric analyses of myelinated and unmyelinated fibers in regenerating sciatic nerves of PNT-1 transgenic and wild-type mice. (A) Diameters of myelinated axons in unoperated and 3-, 5-, and 9-week postlesion sciatic nerves of wild-type (light gray) and transgenic (dark gray) mice. Boxes comprise the 25 and 75 percentiles, horizontal lines within boxes indicate the median, and smaller horizontal lines denote the highest and lowest values. Statistical comparisons between medians were made with an unpaired, nonparametric test (Mann–Whitney). ∗∗∗, P < 0.001; ∗∗, P < 0.01 (22). (B) Ratio of operated to intact axon diameters of the median (squares) and 75 percentile (circles) values in transgenic (solid) and wild-type (open) sciatic nerves at different times after lesion. (C) Size–frequency histogram of unmyelinated fibers from unoperated sciatic nerves (stippled line), and from lesioned wild-type (hatched line) and lesioned transgenic (solid line) sciatic nerves 5 weeks after lesion.

Figure 3

Analyses of target reinnervation. The density of axonal bundles was estimated in semi-thin sections of foot pad dermis (Left) and flexor plantaris muscle (Right). In the upper graphs, the density of axonal bundles per 10,000 μm² is indicated for unoperated nerves (control), and operated transgenic (PNT-1) and wild-type mice 5 weeks after nerve injury (n = 4). The lower micrographs show examples of semi-thin sections of dermis and muscle. Axonal bundles are indicated by arrows. (Bar = 10 μm.)

Figure 4

Functional regeneration of motor and sensory axons in sciatic nerves of PNT-1 transgenic and wild-type mice. (A) Schematic representation of the strategy used for electrophysiological recordings of CAPs in dorsal and ventral roots. The total area of CAPs obtained after proximal stimulation was set to 100%. (B) Relative CAPs in lesioned motor (Upper) and sensory (Lower) axons of PNT-1 transgenic (solid circles) and wild-type (open circles) mice with a 5-mm (Left) or a 10-mm (Right) nerve bypass (n = 3; mean ± SEM).

Figure 5

Time course of skeletal muscle weight decay in PNT-1 transgenic and wild-type mice. Gastrocnemius muscle weights of transgenic (solid circles) and wild-type (open circles) mice were determined 2, 3, 4, and 10 weeks after sciatic nerve transection (10-mm nerve bypass). Values are expressed as percentage of the weight of the contralateral muscle (n = 3; mean ± SEM).