Leads to transcriptional, biochemical and morphological changes in astrocytes termed reactive astrogliosis. [7] The signaling cues leading to this damage are only partly known, but appear to be influenced by the cause of damage. [8] The resulting glial scar is widely considered to have a negative impact on mechanisms of recovery. [9] However, positive aspects of reactive astrocytes have also been shown. [10] S1P could be a direct mediator of reactive gliosis via activation of specific G protein-coupled S1P receptors, S1PR1?. [11,12] Some recent reports suggest that S1P and 10457188 the S1P receptor agonist FTYFTY720 Enhances Recovery in Photothrombotic Strokeinfluences glial scarring in experimental autoimmune encephalitis and spinal cord injury. [13,14]. We examined whether behavioral recovery could be pharmacologically enhanced by delayed administration of FTY720 in a model of stroke assessing functional outcome over 31 days, astrocytic reactivity, synaptic morphology and as a possible mechanism of recovery, the influence of FTY720-treatment on the expression of neurotrophic factors. We furthermore studied the concentrations of S1P, FTY720 and phospho-FTY720 (pFTY720) and the expression levels of key enzymes of S1P metabolism in the periinfarct cortex.treatment with i.p. FTY720 (Selleck Chemicals) 1 mg/kg b.i.d. for 5 days versus 0.9 saline was started.Behavioural AnalysisAnalysis of the behavioural outcome after PT was performed as described previously. [15] The video analysis was done by an examiner blinded to the treatment groups. The GWT was performed in a cage with an area of 600 cm2. The bottom was replaced by a mesh with an opening width of 1 cm2. The cage was placed at a height of 20 cm. A mirror placed under the cage allowed recording the mice walking on the grid on video for 5 min. The total number of steps was counted, whereas one step is defined as the movement of all four limbs. Furthermore the foot faults of the left paretic forepaw were counted. A foot fault is defined by a limb going through the grid or the paw resting on the grid only with its wrist. For the CT, animals were placed in a plexiglas cylinder. While mice explored the surface by rearing up on their hindlimbs, the time of wall placement was recorded for the right forelimb, left forelimb and both forelimbs simultaneously. The difference between paretic (left) and non-paretic (right) plus bilateral placement was evaluated for each mouse.Methods Animals and Experimental Model of Photothrombotic StrokeMale C57BL/6 mice (6?2 weeks old, strain J) were used in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1996). All animal experiments were approved by the local government authorities (Regierungspraesidium Darmstadt). Stroke was induced by photothrombosis (PT) as described previously. [15] Briefly, after injection of buprenorphine, inhalative anesthesia using 2 isoflurane was performed. A cold light source (KL1500, LCD, Zeiss, Jena, Germany) was connected to a 406 objective, resulting in a 3 mm diameter light beam. The beam was stereotactically placed 1.5 mm lateral to the bregma. 5 minutes after the injection of 0.2 ml rose-bengal (Sigma-Aldrich, Taufkirchen, Germany; 10 mg/ml), the scull of the animal was illuminated for 15 minutes, inducing a focal stroke within the animal’s right-hemispheric motor cortex. At the indicated time points, animals were killed using an overdose of isofl.
