The Effect of Centella Asiatica (L.) Urban on an Organotypic Spinal Cord Model

Abstract

Centella asiatica (L.) Urban (CA) is a well- known plant used to improve brain and memory functions in Chinese and Malay traditional medicine. Scientifically it was proven to show neurogenic effect on neural cell lines and in rat’s hippocampus. Its effect on spinal cord (SC) neurons, however, have not been studied widely despite the devastating paralytic effect of SC injury (SCI) and modern medicine’s absence of a cure. Aims of this study are to develop an organotypic model (OM) of the SCI (OMSC) and to investigate neurotrophic effects of a raw extract of CA (RECA) on neurite outgrowths in the model. OMSC was prepared using SC slices obtained by slicing rat pup SC with a vibratome and embedment of SC in a suitable gel block. To achieve this, density of the gel must be as equivalent to the density of the SC, the blade vibration (BV) speed and amplitude must be optimised and thickness of the slice produced must maintain viability in culture. After optimisation, the optimum parameters were; use postnatal day 8 rat pups embedded in 10% gelatine gel, sliced with BV amplitude of 10 and frequency of 1 to produce 300 µm thick slices. These slices were 100% viable for 8 days in culture. RECA, in concentrations of 0, 100, 200 400 and 800 µg/ml was added to the OMSC media for 7 days, followed by immunostaining for TUJ-1 and GFAP and the image was captured with fluorescence microscope. The investigated parameters were mean neurite count, mean neurite length, mean longest neurite and growth ratio. The tested RECA concentrations showed no cytotoxicity. Anova and Kruskal-Wallis tests showed no significant difference between groups. This may be due to low content of neurotrophic bioactive compounds content in the extract, which probably due to differences in geographical location and extraction method and absence of Nerve Growth Factor (NGF) in the media. Overall, OMSCI was successfully developed using a vibratome and embedding gel as an alternative culture model for testing neurogenesis. However, the tested RECA concentration showed no neurotrophic effects on the spinal cord.