OBJECTIVETo detect the expression of telomerase in glial scar and its correlation with glial scar.

METHODSThere were 120 Sprague Dawley rats were randomly divided into non-interference group of telomerase, interference group of telomerase and control group. Non-interference group and interference group were for spinal cord injury, which adopted Allen's Weight Dropping to make molding; control group was for sham operation to open the vertebral plate and expose spinal marrow, in which spinal cord injury would not be caused. The expression of telomerase and glial fibrillary acidic profein (GFAP) was detected by PCR-ELISA and Western blot, and the formation of glial scar was observed by immunofluorescence on the 1st, 3rd, 5th, 7th, 14th, 28th, 42 th and 56th day after the spinal injury, and analyzed its relativity.

RESULTSThe expression of telomerase in non-interference group was (0.180 ± 0.004 - 1.217 ± 0.072), which was significantly higher than those in interference group (0.028 ± 0.007 - 0.092 ± 0.004, χ(2) = 28.753 - 37.518, P < 0.05) and control group (0.072 ± 0.007 - 0.075 ± 0.004, χ(2) = 18.618 - 41.093, P < 0.05) at all the time, with statistical significance. The expression of GFAP in non-interference group was (1.98 ± 0.15 - 19.40 ± 0.55) which was significantly higher than those in interference group (1.10 ± 0.13 - 16.64 ± 1.02, χ(2) = 14.538 - 37.366, P < 0.05) and control group (0.44 ± 0.05 - 0.48 ± 0.04, χ(2) = 16.733 - 34.041, P < 0.05) at all the time, with statistical significance. The expression of GFAP showed a linear correlation with that of telomerase in non-interference group, and with statistical differences (r = 0.755, P < 0.01). The expression of telomerase in interference group and control group were always negative. Glial scar observed by immunofluorescence in non-interference group was heavier than that in interference group, and control group showed no formation of glial scar.

CONCLUSIONSTelomerase shows a dynamic expression in glial scar and has positive correlational linear relationship with GFAP which shows the formation of glial scar. And the telomerase may be an important factor in promoting the formation of glial scar.

Animals ; Cicatrix ; enzymology ; Glial Fibrillary Acidic Protein ; metabolism ; Neuroglia ; enzymology ; pathology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; enzymology ; pathology ; Telomerase ; metabolism

A subset of patients of amyotrophic lateral sclerosis (ALS) present with mutation of Cu/Zn superoxide dismutase 1 (SOD1), and such mutants caused an ALS-like disorder when expressed in rodents. These findings implicated SOD1 in ALS pathogenesis and made the transgenic animals a widely used ALS model. However, previous studies of these animals have focused largely on motor neuron damage. We report herein that the spinal cords of mice expressing a human SOD1 mutant (hSOD1-G93A), besides showing typical destruction of motor neurons and axons, exhibit significant damage in the sensory system, including Wallerian-like degeneration in axons of dorsal root and dorsal funiculus, and mitochondrial damage in dorsal root ganglia neurons. Thus, hSOD1-G93A mutation causes both motor and sensory neuropathies, and as such the disease developed in the transgenic mice very closely resembles human ALS.
Amyotrophic Lateral Sclerosis/enzymology/*pathology ; Animals ; Axons/*pathology ; Disease Models, Animal ; Ganglia, Spinal/pathology ; Humans ; Mice ; Mice, Transgenic ; Mitochondria/pathology ; Motor Neurons/metabolism/pathology ; Mutation ; Nerve Degeneration/*pathology ; Sensory Receptor Cells/*pathology ; Spinal Cord/*pathology ; Superoxide Dismutase/genetics/*physiology

BACKGROUNDCervical spondylotic myelopathy (CSM), in part, results from degeneration of the posterior longitudinal ligament (PLL), which mechanically compresses the spinal cord. Much research was done on the ossification of PLL, but not concerning the non-ossifying degeneration of cervical PLL. The degeneration of cervical PLL may be related to inflammation. The aim of this study was to elucidate the pathological features of the PLL and the role of cyclooxygenase 2 (COX-2) in the degeneration of the PLL in CSM.

METHODSA total of 23 PLL specimens were collected during surgery from patients with CSM for the histological and immunohistochemical (type II collagen and Ki-67) study. For the control group 14 cervical PLL autopsy specimens were investigated in the same manner. mRNA expression of COX-2 was quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) from 18 PLL specimens of patients with CSM and 18 PLL specimens of autopsy cases. Immunohistochemistry was used to evaluate the cellular location of COX-2 in PLL.

RESULTSA distinct amount of fibrotic area, chondrometaplastic tissue and calcification were found in the PLL of the patient group, compared with the control group. Type II collagen was apparent around chondrometaplastic cells. Ki-67 positive reaction was less than 5%. A COX-2 positive reaction was found in 9 of the patient specimens (39.1%) in which the COX-2 was released from vascular endothelial cells in the PLL. However, such reactions were not found in the control group. Real-time PCR showed that the mRNA expression level of COX-2 in the patient group was significantly higher than that in the control group (P < 0.01).

CONCLUSIONSChondrometaplastic tissue producing type II collagen was identified as the most predominant pathological feature in the degenerative PLL. The higher expression of COX-2 might be related to degeneration of the PLL in CSM.

Adult ; Aged ; Aged, 80 and over ; Cervical Vertebrae ; enzymology ; pathology ; Collagen Type II ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Longitudinal Ligaments ; metabolism ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord Compression ; enzymology ; Spondylosis ; enzymology

OBJECTIVETo investigate Caspase-3 expression and its role in neuronal apoptosis.

METHODSThe T13-L2 spinal cord of rats was injured by traction after the amplitude of P1-N1 wave, monitored by a cortical somatosensory evoked potential (CSEP) monitor, decreased to seventy percent of that before operation. Then rats were killed in 6 h, 1 d, 4 d, 7 d, 14 d and 21 d respectively after operation. Flow cytometer terminal deoxynucleotldyl transferease-mediated biotinylated deoxynuridine triphosphate nick end labeling (TUNEL), Caspase-3 activity assay and immunohistochemical method were applied to investigate Caspase-3 expression in the spinal cord tissue and to study neuronal apoptosis in rats.

RESULTSAfter spinal cord injury, apoptotic cells detected by flow cytometry and TUNEL-positive cells were significantly more, and positive immunohistochemical staining of Caspase-3 and Caspase-3 activity were significantly higher in Group injury than in Groups control and laminectomy, respectively (P > 0.05, P > 0.01). Similar trend of changes was noticed in apoptotic cells, TUNEL-positive cells and positive immunohistochemical staining of Caspase-3, all of which reached their respective peak 7 days after operation. Caspase-3 activity reached its peak, however, 4 days postoperatively.

CONCLUSIONSIncreased expression and activity of Caspase-3 protein in neurons after tractive spinal cord injury is the biochemical signal of early spinal cell apoptosis. It is of great significance for understanding the mechanism of spinal cord injury.

Animals ; Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Flow Cytometry ; Immunohistochemistry ; In Situ Nick-End Labeling ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; enzymology ; pathology

Animals ; Apoptosis ; drug effects ; Enzyme Inhibitors ; pharmacology ; Imidazoles ; pharmacology ; Pyridines ; pharmacology ; Rats ; Signal Transduction ; Spinal Cord Injuries ; enzymology ; pathology ; p38 Mitogen-Activated Protein Kinases ; metabolism

Acute spinal cord injury (SCI) is two-step process that first involves the primary mechanical injury and then the secondary injury is induced by various biochemical reactions. Apoptosis is one of secondary SCI mechanisms and it is thought to play an important role for the delayed neuronal injury. The enhanced formation of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of apoptosis in SCI. The level of .iNOS mRNA peaked at 6 hr after SCI and it declined until 72 hr after SCI in a rat model. Double-immunofluorescence staining revealed that iNOS positive cells were stained for ED-1, synaptophysin, GFAP, and oligodendrocyte marker. The terminal deoxynucleotidyl-transferase-mediated dUDP-biotin nick end-labeling (TUNEL) positive cell count was higher for the 72 hr post-SCI group than for the 24 hr post-SCI group. This cell count was also higher going in the caudal direction than in the rostral direction from the epicenter, and especially for the 72 hr group. Treatment with a selective iNOS inhibitor resulted in the reduction of TUNEL-positive cells at the lesion site. These findings suggest that nitric oxide generated by the iNOS of macrophages, neurons, oligodentrocytes, and astrocytes plays an important role for the acute secondary SCI that results from apoptotic cell death.
Analysis of Variance ; Animals ; Apoptosis ; Comparative Study ; Glial Fibrillary Acidic Protein/analysis ; In Situ Nick-End Labeling ; Microscopy, Fluorescence ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord/chemistry/enzymology/pathology ; Spinal Cord Injuries/*enzymology/pathology/physiopathology ; Time Factors

OBJECTIVETo investigate gene expression of three nitric oxide synthase isozymes in injured spinal cord tissue.

METHODSThirty-six adult SD rats were randomly divided into six groups: a normal group and five injury groups, with six per each group. Animals in the injury groups were sacrificed at 2, 6, 12, 24, 48 h after injury. A compression injury model on the spinal cord was made according to Nystrom B et al and gene expression of the three NOS isozymes were examined by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSGene expression of nNOS and eNOS were detectable in the normal group and were up-regulated quickly after injury, reaching a maximum at 6 h: (0.633 +/- 0.012) and (1.236 +/- 0.207). Gene expression of iNOS was detectable only in the injury groups and it was gradually up-regulated after injury, reaching a maximum at 24 h: (1.043 +/- 0.049).

CONCLUSIONInjury to the spinal cord leads to early up-regulation of cNOS and late up-regulation of iNOS. Different NOS isozymes may play different roles in secondary spinal cord injury.

Animals ; Female ; Gene Expression Regulation, Enzymologic ; Male ; Nitric Oxide Synthase ; genetics ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; RNA ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord ; enzymology ; pathology ; Spinal Cord Injuries ; enzymology ; genetics

OBJECTIVETo determine the effects of recombinant soluble complement receptor type I (sCR1) on the immune inflammatory reaction in acute spinal cord injury tissue of rats and its protective effects.

METHODSSD rat models of acute spinal cord injury were prepared by modified Allen's method. The motor function of the rat lower extremities in sCR1 group and normal saline (NS) group was evaluated by the tiltboard experiment at 12 h, 1 d, 3 d, 7 d, and 14 d. The neutrophil infiltration and C3c positive expression were observed. The myeloperoxidase activity was assessed in the injury tissue at 12 h, 1 d, 3 d, 7 d, and 14 d after injury in the two groups.

RESULTSThe motor function of rat in sCR1 group at 3 d, 7 d, and 14 d was obviously better than that in NS group (P<0.01, P<0.01, P<0.01). C3c positive expression in sCR1 group at each time point after injury was obviously less than that in NS group (P<0.01). The myeloperoxidase activity in sCR1 group at each time point after injury was obviously less than that in NS group (P<0.01).

CONCLUSIONSRecombinant soluble complement receptor type I (sCR1) can lessen the immune inflammatory reaction in acute spinal cord injury tissue and relieve secondary spinal cord injury by inhibiting the activation of the complement system.

Animals ; Disease Models, Animal ; Immunohistochemistry ; Inflammation ; Peroxidase ; biosynthesis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Complement ; therapeutic use ; Recombinant Proteins ; therapeutic use ; Spinal Cord Injuries ; drug therapy ; enzymology ; pathology

To elucidate the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we analyzed the expression of constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) in the spinal cords of rats with EAE. We further examined the structural interaction between apoptotic cells and spinal cord cells including neurons and astrocytes, which are potent cell types of nitric oxide (NO) production in the brain. Western blot analysis showed that three forms of NOS significantly increased in the spinal cords of rats at the peak stage of EAE, while small amounts of these enzymes were identified in the spinal cords of rats without EAE. Immunohistochemical study showed that the expression of either nNOS or eNOS increased in the brain cells including neurons and astrocytes during the peak and recovery stages of EAE, while the expression of iNOS was found mainly in the inflammatory macrophages in the perivascular EAE lesions. Double labeling showed that apoptotic cells had intimate contacts with either neurons or astrocytes, which are major cell types to express nNOS and eNOS constitutively. Our results suggest that the three NOS may play an important role in the recovery of EAE.
Animals ; Apoptosis ; Encephalomyelitis, Autoimmune, Experimental/*enzymology ; Endothelium, Vascular/enzymology ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Nitric Oxide Synthase/*metabolism ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase Type III ; Rats ; Rats, Inbred Lew ; Spinal Cord/*enzymology/pathology

OBJECTIVETo study the effects of Zuogui Pill (, ZGP) and Yougui Pill (, YGP) on the expressions of brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling of axonal regeneration in the Lewis rats with experimental autoimmune encephalomyelitis (EAE), in order to explore the possible mechanism of ZGP and YGP on promoting axonal regeneration.

METHODSThe rats were randomly divided into normal control (NC), model (MO), prednisone acetate (PA), ZGP and YGP groups. The EAE model of rat was established by injecting antigen containing myelin basic protein (MBP)68-86. The brain and spinal cord were harvested on the 14th and 28th day post-immunization (PI), the protein and mRNA expression of BDNF and PKA in the brain and spinal cord of rats were detected by Western blot analysis and real-time quantitative polymerase chain reaction (PCR), and the cAMP levels were detected by using enzyme-immunoassay method.

RESULTS(1) On the 28th day PI, the mRNA expression of BDNF in brain white matter and spinal cord of rats in ZGP and YGP groups were up-regulated, especially in YGP group (P<0.05 or P<0.01). (2) On the 14th day PI, the cAMP levels in brain white matters significantly increased in PA and YGP groups compared with MO group (P<0.05 or P<0.01), and the cAMP level in YGP group was higher than that in ZGP group (P<0.05). The cAMP level in spinal cord also significantly increased in YGP group compared with MO, PA and ZGP groups, respectively (P<0.01). (3) On the 14th day PI, the PKA expression in spinal cord of rats in ZGP group was significantly decreased compared with MO and YGP groups, respectively (P<0.05). (4) On the 28th day PI, there was a positive correlation between cAMP and PKA expression in the brain white matter of YGP rats.

CONCLUSIONSThe results suggest that ZGP and YGP may promote axonal regeneration by modulating cAMP/PKA signal transduction pathway, but the targets of molecular mechanism of ZGP may be different from those of YGP.

Animals ; Axons ; drug effects ; pathology ; Brain ; drug effects ; metabolism ; pathology ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Cyclic AMP ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; enzymology ; genetics ; Female ; Gene Expression Regulation ; drug effects ; Nerve Regeneration ; drug effects ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred Lew ; Signal Transduction ; drug effects ; genetics ; Spinal Cord ; drug effects ; metabolism ; pathology ; Tablets