Role of Telomerase Reverse Transcriptase in Glial

Authors: Xu Tao Yang Mingkun Sheng Weibin Guo Hailong Kan Rui Tu Laiyong

Publish Date: 2013/06/22

Volume: 38, Issue: 9, Pages: 1914-1920

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Abstract

The study aims to determine the expression of telomerase reverse transcriptase TERT in the glial scar following spinal cord injury in the rat and to explore its relationship with glial scar formation A total of 120 Sprague–Dawley rats were randomly divided into three groups SCI only group without TERT interference TERT siRNA group with TERT interference and sham group The TERT siRNA and SCI only groups received spinal cord injury induced by the modified Allen’s weight drop method In the sham group the vertebral plate was opened to expose the spinal cord but no injury was modeled Five rats from each group were sacrificed under anesthesia at days 1 3 5 7 14 28 42 and 56 after spinal cord injury Specimens were removed for observation of glial scar formation using hematoxylineosin staining and immunofluorescence detection mRNA and protein expressions of TERT and glial fibrillary acidic protein GFAP were detected by reversetranscription RTPCR and western blotting respectively Hematoxylineosin staining showed evidence of gliosis and glial scarring in the spinal cord injury zone of the TERT siRNA and SCI only groups but not in the sham group Immunofluorescence detection showed a significant increase in GFAP expression at all time points after spinal cord injury in the SCI only group 81  compared with the TERT siRNA group 67  and sham group 2  In contrast the expression of neurofilament protein 200 NF200 was gradually reduced and remained at a stable level until 28 days in the SCI only group There were no NF200labeled cells in the spinal cord glial scar and cavity at day 56 after spinal cord injury NF200 expression at each time point was significantly lower in the SCI only group than the TERT siRNA group while there was no change in the sham group Western blotting showed that TERT and GFAP protein expressions changed dynamically and showed a linear relationship in the SCI only group r = 0765 P  001 while there was no obvious linear relationship in the sham group r = 0208 P = 0121 RTPCR results showed a dynamic expression of TERT and GFAP mRNA in the SCI only group exhibiting a linear relationship r = 0722 P  001 while there was no linear relationship in the sham group r = 0206 P = 0180 Our data indicate that TERT has a dynamic expression in the spinal cord glial scar which positively correlates to GFAP expression and may be important for promoting glial scar formationSpinal cord injury can induce a series of severe motor sensory and autonomic dysfunctions in humans Formation and development of glial scarring is the predominant pathological change following spinal cord injury 1 Experimental studies have shown that various interventions for spinal cord injury are unable to penetrate the glial scar barrier to prevent glial scar formation Astrocytes play a critical role in glial scar formation and inhibition of axonal regeneration 2 3In addition to protecting the telomere telomerase reverse transcriptase TERT has multiple nontelomere lengthdependent effects that are closely associated with cell activation proliferation and apoptosis 6 7 8 9 Some researchers reported that excitatory and traumatic brain injury could induce telomerase expression in microglia by activating a series of cytokines and growth factors thereby promoting glial scar formation 10 However the role of TERT in the activation and proliferation of astrocytes after spinal cord injury and glial scar formation and development remains unclear Thus in the present study we examined the effects of TERT on glial scar formation after spinal cord injury in ratsThe reverse transcription RTPCR TERT assay kit was purchased from Roche St Louis MO USA The GFAP detection kit FITCconjugated goat antirabbit antibody mouse antiGFAP antibody rabbit antiGFAP antibody and neurofilament protein 200 NF200 were purchased from Sigma The plasmid vector PBCANPSTERT was provided by Invitrogen Carlsbad CA USA The PCR instrument was from BioRad Inc Berkeley CA USAA total of 120 male Sprague–Dawley rats clean grade 180–220 g were purchased from the Experimental Animal Center Xinjiang Medical University license No SYXK Xin 2003001 The rats were randomized into SCI only group TERT siRNA group and sham group n = 40 rats per group All rats were anesthetized with a combination of 2 mL ketamine 1 mL atropine 2 mL diazepam and 5 mL 085  sodium chloride via intraperitoneal injection 05 mL/100 g After anesthesia the modified Allen’s weight drop method was used to induce spinal cord injury at the T8 segment in the TERT siRNA and SCI only groups under aseptic conditions 11 After modeling the bleeding was stopped and the incision sutured and the bladder was manually squeezed every 8 h to help urination until spontaneous voiding In the sham group the spinal cord was exposed but not damaged On the basis of GenBank bioinformatics analysis we designed sense and antisense nucleotides targeting astrocyte TERT mRNA in rats antisense oligodeoxynucleotide 5′GTTAGGGTTAG3 sense oligodeoxynucleotide 5′CTAACCCTAAC3′ In this study we used PBCANPsTERT to effectively inhibit TERT expression In the TERT siRNA group PBCANPsTERT saline solution 91861692 pmol/μL was injected at a dose of 50 μg/kg body mass into the surgical site at 30 min after modeling twice per day No administration was performed in the SCI only groups and sham groupsIn each group five rats were sacrificed under anesthesia at days 1 3 5 7 14 28 42 and 56 Under sterile conditions 50 mg of spinal cord tissue 5 mm in length was taken from the central zone of the injured spinal cord and was then rinsed with 01  DEPC water and stored in nucleasefree cryovials at −80 °C

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