Fig 1: Expression of class 3 Semaphorin family members in mouse cornea and trigeminal ganglia.RT-PCR and qPCR of class 3 Semaphorins was performed in isolated cells and tissues as detailed in Materials and Methods. Immunofluorescence staining was performed in eyes from intact mice (basal control) and mice subjected to corneal epithelium debridement. (A) All class 3 Semaphorins are expressed in the corneal epithelium and TG, with Sema3B and Sema3F showing higher and consistent levels. (B) The gene expression of Sema3A and Sema3F after corneal epithelium debridement significantly changed from their basal level. While Sema3F expression in the corneal epithelium is reduced to 50% after one day post debridement and slowly recovers to normal levels after 2 weeks, Sema3A is sharply upregulated soon after injury and remains elevated over the entire evaluated period. Values represent mean ± SD, experiments were performed in triplicate (for each experiment tissues were collected from n = 4 mice, * indicates p< 0.05 vs day 0). (C) Eye crossections stained with anti Sema3A (red) antibody and DAPI (blue, nuclear staining) shows apical expression of Sema3A in basal control. Upon injury there is high expression of Sema3A in the basal layers of the epithelium. Dashed white-square in left image shows the area of analysis. Images are representative of n = 4 mice.
Fig 2: Sema3A does not inhibit the NGF induced neuronal growth of adult DRG and TG neurons.Isolated DRG and TG neurons were incubated with NGF for 3 and 2 days respectively to induce neuronal growth and then treated with different concentrations of Sema3A to determine any significant growth inhibitory effect on these PNS neurons. Neurite growth was evaluated 24 h after addition of Sema3A. (A) We found that about one third of the isolated neurons responded to the NGF treatment and addition of Sema3A produced no inhibitory effects such as the neurons kept growing similarly as compared to control cells treated with NGF alone. (B) TG neurons responded to NGF similarly to DRG neurons and addition of Sema3A did not alter the growth of neurites and no axonal retraction was observed. Neuronal growth was similar to controls that were resupplied with NGF over the course of the experiment. Values represent mean ± SEM, experiments were performed in triplicate, each dish had an average of 200 neurons for DRG and 60 neurons for TG; all neurons in every dish were evaluated.
Fig 3: Sema3A is a potent inducer of neuronal growth.Since Sema3A is highly expressed in the cornea upon injury and does not inhibit the NGF induced growth on adult PNS neurons, we evaluated the effects of adding Sema3A alone to cultured neurons and compared to the NGF induced growth. (A) As described above DRG neurons responded well to NGF treatment. Surprisingly, Sema3A by itself is also a potent inducer of neuronal growth and similar neurite extension was observed at doses of 20 ng/ml Sema3A or higher. (B) Similarly, Sema3A is also a potent inducer of neuronal growth on TG neurons, and the number of TG neurons showing neurite growth was similar to that of neurons treated with NGF alone when incubated with Sema3A at 10 ng/ml or higher. (C) Sema3A from different sources, recombinant human or mouse, induced equally strong neuronal growth of TG neurons at equal concentrations. Values represent mean ± SD, experiments were performed in triplicate, each dish had an average of 200 neurons for DRG and 60 neurons for TG, and all neurons in a dish were evaluated. Neurite growth was evaluated at day 4 for DRG neurons and at day 3 for TG neurons. * indicate p<0.05 vs NGF. (R&D = from R&D Systems, m = murine, h = human, SB = Sino Biological Inc.).
Fig 4: Sema3A induction of neuronal growth is comparable to NGF.The neuronal promoting effects of Sema3A and NGF were compared side by side in adult TG and DRG neurons. Neurons were treated either with growth medium alone as negative control or with 50ng/ml NGF or Sema3A. (A) The length of the neurites was quantified after 48 and 72 h post treatment and expressed as percentage of the total neurons in the dish. In TG neurons, Sema3A induced higher percentage of cells with neurites, however quantification shows that this effect was not statistically significant. In DRG neurons the percentage of short, medium or long neurites was comparable between Sema3A and NGF treatment. (B) The average length and number of branches of long neurites was compared and significant differences observed when compared to untreated control neurons, but similar effects observed between Sema3A and NGF treatments. Values represent mean ± SD, experiments were performed in triplicate, each dish had an average of 200 neurons for DRG and 60 neurons for TG; all neurons in every dish were evaluated. * indicates p<0.05 vs Control.
Fig 5: Sema3A induce nerve regeneration in injured corneas.The neuronal promoting effects of Sema3A were tested on thy1-YFP mice subjected to superficial corneal epithelial debridement. The debridement of the epithelium also removes the sub basal nerve plexus without affecting the cornea nerves in the stroma. Insertion of an intrastromal pellet containing Sema3A or vehicle (see Material and methods) allows for the slow release into the cornea. (A) Pellets containing vehicle (PBS) induced a discrete growth of sub basal nerves into the injured area. (B) However, addition of Sema3A induced faster regeneration of the superficial corneal nerves and higher nerve density was observed. (C) Quantification of nerve regeneration in the corneal injured area shows that Sema3A induced 3 folds higher nerve regeneration than control mice. White arrows = superficial nerves, yellow arrows = pellet. Values represent mean ± SEM, experiments were performed in triplicate, n = 5 per treatment, * indicates p<0.05 by ANOVA.
Supplier Page from Sino Biological, Inc. for Mouse Semaphorin 3A / SEMA3A Protein (Fc Tag)