Changes in Neurons, Glia and Extracellular Matrix Molecules after Spinal Cord Hemisection in Tenascin-C deficient Mice

Abstract

Patients suffering from spinal cord (SC) injury are challenged to maintain an acceptable quality of life and complete functional recovery after SC injury has not been described to date. Tenascin C (TNC), an extracellular matrix (ECM) glycoprotein is involved in wound healing and its role is somewhat paradoxical, because in vitro it can be both neurite-outgrowth promoting and repellent. To assess its effects in vivo, 20 TNC-/- knock out (TNC-/-) and 20 TNC+/+ wild type (TNC+/+) mice underwent lumbar SC hemisection. According to the sequence of the inflammatory response, systemic perfusion was performed at 1, 3, 7 and 14 days post surgery. Cryostat sections were examined in H&E and combined Masson trichrome and Verhoeff stain as well as by immunohistochemistry using antibodies against neurofilament (NF), GFAP (astrocytes), F4/80 (microglia) and the ECM molecules collagen type IV, laminin and fibronectin. Staining intensity was quantified by measuring Integrated Optical Density at the incision site, the surrounding area and distant from the lesion site. TNC+/+ mice resembled the findings of previous studies in normal mice. However, distinct alterations after SC hemisection were seen in TNC-/- mice. After one day, an early onset lymphocytic infiltration was seen in TNC-/- mice. Seven days after injury neutrophil influx was more prominent in TNC-/- mice compared to TNC+/+ mice. Quantitative densitometry demonstrated that microglial and astrocytic responses to injury were identical in TNC-/- and TNC+/+ mice. However, after fourteen days, fibronectin was significantly (p=0.036) decreased in TNC-/- mice. At the same time, NF-positive axonal sprouts were markedly increased along the incision edges of TNC-/- mice.

The increase in NF-positive sprouts in TNC-/- mice indicates that lack of TNC fundamentally alters ECM composition in a way that axonal fibres are more likely to penetrate SC scar tissue. These differences between TNC+/+ and TNC-/- mice were detected fourteen days after injury. Hence further in vivo studies should be carried out at later time points in order to assess the beneficial effect of TNC depletion in axonal regeneration further.