Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision

Abstract

Nanotechnology is often associated with materials fabrication, microelectronics, and microfluidics. Until now, the use of nanotechnology and molecular self assembly in biomedicine to repair injured brain structures has not been explored. To achieve axonal regeneration after injury in the CNS, several formidable barriers must be overcome, such as scar tissue formation after tissue injury, gaps in nervous tissue formed during phagocytosis of dying cells after injury, and the failure of many adult neurons to initiate axonal extension. Using the mammalian visual system as a model, we report that a designed self-assembling peptide nanofiber scaffold creates a permissive environment for axons not only to regenerate through the site of an acute injury but also to knit the brain tissue together. In experiments using a severed optic tract in the hamster, we show that regenerated axons reconnect to target tissues with sufficient density to promote functional return of vision, as evidenced by visually elicited orienting behavior. The peptide nanofiber scaffold not only represents a previously undiscovered nanobiomedical technology for tissue repair and restoration but also raises the possibility of effective treatment of CNS and other tissue or organ trauma.

Fig. 1.

SAPNS repair for the animal brain. (a) Molecular model of the RADA16-I molecular building block. (b) Molecular model of numerous RADA16-I molecules undergo self assembly to form well ordered nanofibers with the hydrophobic alanine sandwich inside and hydrophilic residues on the outside. (c) The SAPNS is examined by using scanning electron microscopy. (Scale bar, 500 nm.)

Fig. 2.

SAPNS heals the brain in young animals. (a) Dorsal view reconstruction of the hamster brain with cortex removed. Rostral is to the left, and caudal is to the right. The blue line depicts the location of the optic tract transection made at P2 in the SC of the midbrain. Also shown: pretectal area (PT), lateral posterior nucleus (LP), medial geniculate body (MGB), lateral geniculate body (LGB), and inferior colliculus (IC). (b) Schematic illustration of a parasagittal section of the midbrain of a hamster, with the position and depth of the surgical knife cut indicated in the SC. Rostral is always to the left. Animals received an injection into the cut of 10 μl of saline in the controls or 10 μl of 1% SAPNS. The dark-field composite photos are parasagittal sections from animals 1, 30, and 60 days after lesion and treatment. Arrows indicate the path and extent of knife cut. Animals killed at 24 h after lesion and treatment: saline control animal (c) and SAPNS treatment (d). The treated case (d) has a very small gap, and the surface of the tissue has already started to reconnect both sides. (e and f) Thirty–day postlesion cases; e is a saline control, and f is an example of SAPNS-treated animals. Note the large gap in e. In the SAPNS treatment case (f), the gap is completely gone, and tissue has reconnected across the injury site. (g) Dark-field composite photo of a 60-day post-SAPNS solution-treated animal. (h) A corresponding bright-field picture. Note the lack of tissue disruption in the bright-field composite picture. All SAPNS solution-treated cases appear to have reconnected at the site of the lesion. [Scale bars, 500 μm (b); 100 μm (c–h).]

Fig. 3.

SAPNS allows axons to regenerate through the lesion site in brain. The dark-field composite photos are parasagittal sections from animals 30 days after lesion and treatment. (a) Section from brain of 30-day-old hamster with 10 μl of saline injected in the lesion at P2. The cavity shows the failure of the tissue healing. The retinal projections, in light green at the top left edge of the cavity, have stopped and did not cross the lesion. Arrows indicate path and extent of knife cut. (b) A similar section from a 30-day-old hamster with a P2 lesion injected with 10 μl of 1% SAPNS. The site of the lesion has healed, and axons have grown through the treated area and reached the caudal part of the SC. Axons from the retina are indicated by light-green fluorescence. The boxed area is an area of dense termination of axons that have crossed the lesion. Arrows indicate path and extent of knife cut. (c) Enlarged view of boxed area in b. The regrown axons, shown in white, were traced with cholera-toxin fragment B labeling by using immunohistochemistry for amplification of the tracer. (Scale bars, 100 μm.)

Fig. 4.

Optic tract (OT) regenerated through lesion in adults. (a) Dorsal-view reconstruction of the hamster brain with cortex removed, as in Fig. 2a. Rostral is to the left, and caudal is to the right in all figures. The red line depicts the location of the OT transection at the brachium of the SC made at 8 weeks. (b and f) Schematic illustrations of parasagittal sections of the midbrain of a hamster, with the position and depth of the surgical knife cut indicated in the brachium of the SC. Animals received an injection of 30 μl of saline in the controls or 30 μl of 1% SAPNS solution. Two boxes in f show the locations of the dark-field pictures (c and e). (c) Dark-field photo of a parasagittal section from the brain of an 8-month old hamster treated with SAPNS at the time of surgery in the lesion site. The yellow dots show the location of the lesion. The axons, shown by green fluorescence, have grown through the site of lesion and are reinnervating the SC. Note the lack of tissue disruption. The boxed area is enlarged in d. Dense regenerated axons, in green, have grown through the lesion site. The yellow dots mark the location where the lesion was made and the area of subsequent treatment. (e) Dark-field picture from the superficial layers of SC caudal to the lesion, at a higher magnification; shown are regenerated axons and their dense terminals (in white). (g) Dark-field photo of a parasagittal section from the brain of an 8-month-old hamster treated with SAPNS at the time of surgery (at age 8 wk) in the lesion site. The axons, shown by green fluorescence, have grown through the site of lesion and are reinnervating the SC at ≈82% of normal density. Note the axon tracts in the deep layers of the SC. [Scale bars, 500 μm (b and f), 50 μm (c–e).]

Fig. 5.

Optic tract regeneration and functional return of vision. This SAPNS-treated adult animal turns toward the stimulus in the affected right visual field in small steps, prolonged here by movements of the stimulus away from the animal. Each frame is taken from a single turning movement, at times 0.00 (a), 0.27 (b), 0.53 (c), and 0.80 (d) sec from movement initiation. The animal reached the stimulus in the last frame. This is 29% slower than most turns by a normal animal. The recording was made 6 weeks after surgery and treatment when the animal started to show a response. See Movies 1 and 2, which are published as supporting information on the PNAS web site, for regenerated and control animals. (e) The graph shows the frequency (in percent) of response of all animals when a visual stimulus was presented to the eye connected to the treated optic tract (blue) or spontaneous turns of blind animals of the control group (yellow). The exceptions were the two animals that lost their SC after surgery; they were included in the average of the controls, because there was no difference in their response compared with the controls. On the x axis is the session number. Testing started 6 weeks after surgery (error bars SEM).