Developing a trans-multisynaptic tracer to map the neural circuit of recovered sciatic nerve after treatment with nerve growth factor

Abstract

Nerve growth factor (NGF) has been shown to support the survival and differentiation of neurons. In this study, we first developed a retrograde trans-multisynaptic tracer PRV580 expressing the mCherry fluorescent protein based on pseudorabies virus Bartha strain to map the neural circuit of sciatic nerve. Secondly, the newly developed PRV580 was used to map the neural circuit of the recovering sciatic nerve upon treatment with NGF. Our results showed that red signals from PRV580 were observed in various brain regions. Among these regions, many areas of the pyramidal system and the extra-pyramidal system had been mapped, accounting for as much as 56.8 % of the total inputs. Furthermore, we found that NGF could significantly increase the ratio of total input (29.05 %) compared to PBS (3.65 %), indicating that NGF indeed can aid in the repair of injured sciatic nerve. These findings indicated that NGF has therapeutic ability for the treatment of peripheral nerve injuries and virus-based tracers can be used to monitor the recovery.

Keywords: Nerve growth factor; Neural circuit; Retrograde trans-multisynaptic tracer; Sciatic nerve.

Fig. 1

Preparation of recombinant PRV580. Cloning diagram of recombinant PRV580. The top represents PRV Bartha genome. The middle represents the expression cassette of plasmid PS580, which is flanked by left arm left homologous arm (left arm) and right homologous arm (right arm). The bottom represents the genome of recombinant PRV580. (B) Purification of recombinant PRV580 infects BHK21 cell. (C) The plaque assay was performed on BHK21 cells. (D) The growth curve of PRV580 and its parent virus. Virus infects BHK21 at moi=0.1 and the sample was collected at indicated time points (12, 24, 36, 48 and 60 hpi).

Fig. 2

The sciatic nerve pathway was mapped using reterograde trans-multisynaptic tracer PRV580. The PRV580 (1.5 × 10¹⁰ PFU/ml) was injected into the sciatic nerve of rat. The slices were stained with DAPI and imaged. The abbreviations of these labelled brain regions are as follow: medial preoptic nucleus (MPN), lateral preoptic area (LPO), periaqueductal gray (PAG), ventral tegmental area (VTA), medial hypothalamic nucleus (MH), lateral hypothalamic area (LH), pontine reticular nucleus (PRN), reticulotegmental nucleus of the pons (RtTg), lateral lemniscus (LL), medial vestibular nucleus (MVN), gigantocellular reticular nucleus (GRN), mesencephalic reticular formation (mRt), isthmic reticular formation (isRt), reticular nucleus (RN). In addition, the Tg region includes SPTg, PPTg, LDTg, and VTg.

Fig. 3

The percentage of total inputs of the sciatic nerve pathway. The PRV580 (1.5 ×10¹⁰ PFU/ml) was separately injected into the sciatic nerve of three rats. To determine the percentage of total inputs of different brain regions for each animal, one third of all slices from each animal were selected for data analyzing. The percentage of total input is calculated as the number of red input neurons of each brain region divided by the number of all red neurons from all counted brain slices.

Fig. 4

mNGF can help the injured nerve to repair. (A) The total recovery ratio of the injured sciatic nerve with the help PBS and NGF, respectively. The sciatic nerves of three groups (each group has three rats) were treated with three strategies. PBS-treatment group: the sciatic nerve was completely cut, then sutured, followed with PBS treatment. NGF-treatment group: the sciatic nerve was sutured after transection, and then treated with 10 mg mNGF every day for three weeks. Wild type group: the sciatic nerve was not cut and without any treated. (B) The representative images in GRN, MVN and Tg regions. The labelled input neurons by PRV580 were counted in GRN (C), MVN (D) and Tg (E) in PBS, NGF and wild type groups. The t-test was conducted to compare the difference of recovery ability of the NGF and PBS. Significant differences between pairs are indicated by the p-value. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 5

An overview of the repair ability of NGF on injured sciatic nerve. The sciatic nerves of three groups (each group has three rats) were treated with three strategies. PBS group: the sciatic nerve was completely cut, then sutured, followed with PBS treatment. NGF group: the sciatic nerve was sutured after transection, and then treated with 10 mg mNGF every day for three weeks. Wild type group: the sciatic nerve was not cut and without any treated. The labelled input neurons by PRV580 were counted in different brain regions in PBS, NGF and wild type groups.