AR-A014418 as a glycogen synthase kinase-3 inhibitor: anti-apoptotic and therapeutic potential in experimental spinal cord injury.

OBJECTIVES: We aimed to investigate the effects of AR-A014418, a strong inhibitor specific to GSK-3beta, on neuronal apoptosis and neuroprotection in the traumatic SCI model. MATERIALS AND METHODS: In this study, three groups were generated from 36 Wistar rats; (1) control, (2) spinal cord trauma group created by clip compression technique after laminectomy, and (3) AR-A014418 (4mg/kg, i.p., DMSO) treatment group after laminectomy and spinal cord trauma. The TUNEL assay for apoptosis detection, immunohistochemical staining for bax and TGF-beta were applied in spinal cord tissues. For light microscopic examination, necrotic, and apoptotic cells were counted, and PMNL counting was applied to detect inflammation. Functional recovery was tested by field locomotor test in the 3rd and 7th days following surgery. RESULTS: In the trauma group, diffuse hemorrhage, cavitation, necrosis and edematous regions, degeneration in motor neurons and leukocyte infiltration were observed in gray matter. In the AR-A014418-treated groups, healthy cells were observed in more places compared to the trauma groups, however, cavitation, hemorrhagic, and edematous areas were seen in gray matter. In the AR-A014418-treatment groups, the number of apoptotic cells in the 3rd and 7th days (respectively; p<0.05, p<0.01), were significantly decreased compared to the trauma groups, as were the levels of bax (p<0.01) and TGF-beta 1 immunoreactivity. Results of the locomotor test were significantly increased in the treatment group (p<0.001) as compared to the trauma group. CONCLUSIONS: In this experimental spinal cord trauma model study neural apoptosis was significantly triggered in secondary damage developed after trauma, however, neurological healing was expedited by preventing mitochondrial apoptosis and reducing the inflammation by the potent inhibitor AR-A014418, which is GSK-3beta selective.

Quantitative analysis of cyclooxygenase 2 in the posterior longitudinal ligament of cervical spondylotic myelopathy.

BACKGROUND: Cervical 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. METHODS: A 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. RESULTS: A 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). CONCLUSIONS: Chondrometaplastic 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.

The human G93A-SOD1 mutation in a pre-symptomatic rat model of amyotrophic lateral sclerosis increases the vulnerability to a mild spinal cord compression.

BACKGROUND: Traumatic injuries can undermine neurological functions and act as risk factors for the development of irreversible and fatal neurodegenerative disorders like amyotrophic lateral sclerosis (ALS). In this study, we have investigated how a mutation of the superoxide dismutase 1 (SOD1) gene, linked to the development of ALS, modifies the acute response to a gentle mechanical compression of the spinal cord. In a 7-day post-injury time period, we have performed a comparative ontological analysis of the gene expression profiles of injured spinal cords obtained from pre-symptomatic rats over-expressing the G93A-SOD1 gene mutation and from wild type (WT) littermates. RESULTS: The steady post-injury functional recovery observed in WT rats was accompanied by the early activation at the epicenter of injury of several growth-promoting signals and by the down-regulation of intermediate neurofilaments and of genes involved in the regulation of ion currents at the 7 day post-injury time point. The poor functional recovery observed in G93A-SOD1 transgenic animals was accompanied by the induction of fewer pro-survival signals, by an early activation of inflammatory markers, of several pro-apoptotic genes involved in cytochrome-C release and by the persistent up-regulation of the heavy neurofilament subunits and of genes involved in membrane excitability. These molecular changes occurred along with a pronounced atrophy of spinal cord motor neurones in the G93A-SOD1 rats compared to WT littermates after compression injury. CONCLUSIONS: In an experimental paradigm of mild mechanical trauma which causes no major tissue damage, the G93A-SOD1 gene mutation alters the balance between pro-apoptotic and pro-survival molecular signals in the spinal cord tissue from the pre-symptomatic rat, leading to a premature activation of molecular pathways implicated in the natural development of ALS.

Activation of Ca2+/calmodulin-dependent protein kinase II alpha in the spinal cords of rats with clip compression injury.

Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) is abundant in the central nervous system, where it plays important roles in regulating neuronal plasticity and survival. However, the role of CaMKIIalpha activation in traumatically injured spinal cords remains unclear. This study examined the effects of clip compression injury on levels of phosphorylated CaMKIIalpha (pCaMKIIalpha) and its cellular localization in rat spinal cords. Western blot analysis showed that the pCaMKIIalpha levels in both rostral (days 7, 14, and 21 post-injury) and caudal (days 4, 7, 14, and 21 post-injury) areas of the injury site were more than twice the levels in the non-injured controls. Immunohistochemical examination revealed constitutive localization of pCaMKIIalpha in the superficial lamina of the dorsal horn and neurons in normal spinal cord controls. After spinal cord injury, levels of the same components were markedly increased in both rostral and caudal regions approximately 3 mm from the center of the spinal cord lesions. However, pCaMKIIalpha was very rare in inflammatory cells in the injured spinal cords. In this animal model, CaMKIIalpha may play an important role in the spontaneous reversal of spinal cord dysfunction, thus restoring locomotor activity, possibly by functioning in the reconstruction of synaptic transmission and in protecting neurons from spinal cord injury.

Expression of apoptosis signal-regulating kinase 1 in mouse spinal cord under chronic mechanical compression: possible involvement of the stress-activated mitogen-activated protein kinase pathways in spinal cord cell apoptosis.

STUDY DESIGN: To examine apoptosis signal cascade in neurons and oligodendrocytes under the chronic spinal cord compression of tiptoe-walking Yoshimura (TWY) mouse, which is model of progressive cervical cord compression. OBJECTIVE: To clarify the biologic mechanisms of apoptosis, which may produce destructive changes in the spinal cord under chronic mechanical compression, with a resulting irreversible neurologic deficit. SUMMARY OF BACKGROUND DATA: The stress-activated mitogen-activated protein kinase pathways including ASK1 transmitted apoptosis signals after acute spinal cord injury. Apoptosis in acute spinal cord injury induced both secondary degeneration around the site of injury and chronic demyelination. Chronic spinal cord compression showed myelin destruction, loss of axons, and oligodendrocytes in white matter, and loss of neurons in gray matter. Apoptosis associated with chronic spinal cord compression contributes to these changes. However, the biologic mechanisms of apoptosis in the spinal cord under chronic mechanical compression remain unclear. METHODS: We examined the expression of phosphorylated-apoptosis signal-regulating kinase 1 (ASK1), phosphorylated-c-Jun N-terminal kinase (JNK), phosphorylated-p38 mitogen-activated protein kinase (p38), and activated caspase-3 immunohistologically in TWY mice, an animal model of progressive cervical spinal cord compression, since the ASK1-JNK and -p38 signaling cascades participate in the signaling pathway leading to apoptosis in neural tissue and neuronal culture. RESULTS: Double immunohistochemistry for phosphorylated-ASK1, phosphorylated-JNK, phosphorylated-p38, activated-caspase3, and cell-specific markers confirmed the presence of apoptosis signals in both neurons and oligodendrocytes in compressed spinal cord cells. CONCLUSION: We found that mitogen-activated protein kinase pathways including ASK1, JNK, and p38 were activated in destructive spinal cord under chronic compression.

Upregulation of phospholipase D1 in the spinal cords of rats with clip compression injury.

This study examined phospholipase D 1 (PLD1) expression in the central nervous system following clip compression spinal cord injury (SCI) in Sprague-Dawley rats. After inducing SCI with a vascular clip, the expression of PLD1 in the affected spinal cord was analyzed by Western blot and immunohistochemistry. Western blot analysis showed that the expression of PLD1 gradually increased in the spinal cord on days 0.5, 1, 2, and 4 post injury. Immunohistochemistry showed that some cells, including neurons, astrocytes, and some inflammatory cells, were positive for PLD1 in the lesions at days 1 and 2 post injury. At day 4, the number of PLD1-positive cells in SCI lesions increased, largely matching the increases in ED1-positive macrophages and glial fibrillary acidic protein-positive astrocytes. At this time, macrophages expressed proliferating cell nuclear antigen in addition to PLD1. These results suggest that PLD1 expression is increased in injured spinal cords, and might be involved in the activation and proliferation of macrophages and astrocytes in SCI.

The role of constitutive nitric oxide synthase in pathogenesis of secondary lesion after spinal cord injury. Preliminary results.

BACKGROUND: Secondary lesion (SL) is an early phenomenon of cellular death following spinal cord injury (SCI). Nitric oxide (NO) could be involved in its pathogenesis. NO is a gaseous metabolite produced by 2 constitutive isoforms of NO synthase (cNOS), constantly active, and by 1 inducible isoform (iNOS), synthesized during inflammation and able to produce large amount of NO. High concentrated NO is toxic for cells; therefore, NO concentration is strictly and finely regulated. We suppose that major inhibitory effect on the iNOS expression is represented by the same physiological concentration of NO, synthesized by cNOS. The aim of this study is to assess the role of the 2 cNOS in pathogenesis of SL after SCI in rat. METHODS: A dorsal SCI has been performed on rats (n=5) by a vascular clip (50 g/mm(2) for 15"). Fifteen minutes after trauma, activity of nNOS and eNOS has been measured (U/mg) in the cervical, dorsal and lumbar segments of spinal cord. Uninjured rats (n=5) served as control group. m-RNA for iNOS in untreated rats (n=2) has been also investigated by Northern blotting. RESULTS: In injured rats nNOS activity has shown a reduction in dorsal and lumbar segments, compared to the control group. eNOS activity, highly variable in the control group, has not been detectable in injured spinal cord. i-NOS mRNA has not been found in spinal cord of uninjured rats. CONCLUSIONS: These results would be in line with our hypothesis and provide the bases for other investigations. New therapeutic strategies for SL prevention, based on the modulation of cNOS, will be evaluated.

Promoting glutathione synthesis after spinal cord trauma decreases secondary damage and promotes retention of function.

The study aimed to 1) quantify oxidative stress in spinal cord after crush injury at T6, 2) determine whether the administration of the procysteine compound L-2-oxothiazolidine-4-carboxylate (OTC) would up-regulate glutathione (GSH) synthesis and decrease oxidative stress, and 3) determine whether decreased oxidative stress results in better tissue and function retention. We demonstrate that spinal cord compression (5 s with a 50 g aneurysm clip) at T6 in rats results in oxidative stress that is extensive (significant increases in oxidative stress seen at C3 and L4) and rapid in onset. Indices of oxidative stress used were GSH content, protein carbonyl content, and inactivation of glutathione reductase. Administration of OTC resulted in a marked decrease in oxidative stress associated with a sparing of white matter at T6 (16+/-1.9% retained in OTC-treated animals vs. less than 1% in saline-treated). Behavioral indices in control, saline-treated, and OTC-treated animals after 6 wk were respectively: angle board scores (59 degrees, 32 degrees, and 42 degrees ), modified Tarlov score (7, 2.4, and 4.1), and Basso-Beattie-Bresnahan score (21, 5.3, and 12.9). We conclude that administration of OTC after spinal cord trauma greatly decreases oxidative stress and allows tissue preservation, thereby enabling otherwise paraplegic animals to locomote.

Effect of graded spinal cord compression on cardiovascular neurons in the rostro-ventro-lateral medulla.

In patients with spinal cord injury, cardiovascular disturbances such as hypotension, bradycardia and autonomic dysreflexia can be directly linked to abnormalities of central autonomic control. To date, the changes in bulbospinal innervation of sympathetic preganglionic neurons after compressive spinal cord injury have not been investigated. Thus, we examined the effect of varying severity of compressive spinal cord injury on neurons of the rostro-ventro-lateral medulla, a nucleus of key importance in cardiovascular control. Adult rats with 20 g, 35 g and 50 g clip compression injuries (n= 18) of the cord at T1 and uninjured controls (n=13) were studied. Neurons in the rostro-ventro-lateral medulla with preserved spinal connections eight weeks after spinal cord injury were identified by retrograde labelling with 4% FluoroGold introduced into the cord at T6. Bulbospinal neurons in the rostro-ventro-lateral medulla were also examined immunocytochemically for the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase. In control rats an average of 451+/-12 rostro-ventrolateral medulla neurons were phenylethanolamine-N-methyltransferase positive. Of these, 213+/-6 projected to the T6 spinal cord. The number of rostro-ventro-lateral medulla neurons retrogradely labelled by FluoroGold decreased as a linear function of severity of spinal cord injury (r= -0.95; P<0.0001). After 50g spinal cord injury at T1, only 7+/-1 rostro-ventro-lateral medulla neurons were labelled by FluoroGold, of which 6+/-1 were phenylethanolamine-N-methyltransferase positive. Moreover, the number of phenylethanolamine-N-methyltransferase positive rostro-ventro-lateral medulla neurons decreased to 361+/-16 after 50 g spinal cord injury. We conclude that compressive spinal cord injury results in disconnection of rostro-ventro-lateral medulla neurons, which project to the thoracic spinal cord, and that these changes vary with the severity of injury. The majority of these axotomized rostro-ventro-lateral medulla neurons maintain their immunopositivity for the adrenaline-synthesizing enzyme phenylethanolamine-N-methyltransferase.

Decreased choline acetyltransferase activity in the murine spinal cord motoneurons under chronic mechanical compression.

The tiptoe-walking Yoshimura (twy) mouse is a model of chronic spinal cord compression caused by ossification of intraspinal ligaments. Choline acetyltransferase (CAT), which is known to be a specific marker of cholinergic neurons, best reflects spinal motoneuron function. Changes in CAT immunoreactivity following chronic spinal cord compression in twy mice were investigated quantitatively in order to elucidate spinal motoneuron functional changes according to the degree and direction of compression. Thirty 24-week-old twy mice were used in this study. They were divided into three groups according to the direction of spinal cord compression (anterior, posterior, and lateral) and the CAT immunoreactivities in whole sections of their upper cervical spinal cords were investigated quantitatively using a fluorescence microphotometry system. The lateral compression group showed histological spinal motoneuron atrophy and loss on the compressed, but not the non-compressed, side. Spinal motoneuron atrophy and loss were observed when the severity of spinal canal stenosis due to the ossified lesion, expressed as the occupation rate, was 30% or more, but the spinal motoneurons appeared normal when it was below 30%. The CAT immunofluorescence intensity of the anterior horn showed a linear negative correlation with the degree of canal stenosis. When the occupation rate was below 20%, the CAT immunofluorescence intensities in the anterior horns of the compression and control groups did not differ significantly. The CAT immunofluorescence intensity of twy mice with occupation rates of 20% or more were significantly lower than that of those with occupation rates below 20%. Furthermore, the CAT immunofluorescence intensity was significantly lower on the compressed than the non-compressed side of the lateral compression group. Thus, our findings indicate that an occupation rate of about 20% may be the critical level for functional changes in the spinal motoneurons.

Involvement of the membrane-bound Na,K ATPase in the evolution of experimental injuries of the spinal cord.

Rat spinal cords have been compressed at the region corresponding to D4-D5 by a 50 mg/cm force. After 5, 30 and 60 min this section has been dissected together with the surrounding cord. The ATPase activity of the homogenates was decreased, suggesting an involvement of this enzyme in the generation of edema.

[AMP deaminase deficiency (myoadenylate deaminase). Disease or syndrome?]. / Le déficit en AMP désaminase (myoadénylate désaminase). Maladie ou syndrome?

Two recent publications have shown the advantage of understanding the deficit in AMP desaminase in rheumatology. On this subject, the authors report 4 cases of deficit in AMP desaminase. The first one includes a semiology made of pain, and stiffening, the second case is discovered in the course of a primary muscular disease. The third case is present during the first stage of a spinal cord compression. The fourth case is a muscular deficit accompanied with a histological picture of inflammation, considered initially as a chronic polymyositis, but explained secondarily as a pseudo-polymyositis form of facio-scapulo-humeral dystrophy. In this respect, the cases from the literature are divided into three groups: asymptomatic, infraclinical forms, forms occurring in the course of specific diseases (muscular diseases, spinal cord diseases, inflammations of the connective tissues, metabolic diseases), apparently isolated forms. In the latter, emerges a semiology made of pain, cramps, stiffening of the lower extremities occurring on exertion. However, the specificity of the symptoms remains to be discussed.

Spinal cord compression by extramedullary hemopoietic tissue in pyruvate-kinase-deficiency-caused hemolytic anemia.

Spinal cord compression by extramedullary hemopoietic tissue is a rare complication of hemolytic anemias. This is the first report of this complication in hemolytic anemia caused by pyruvate-kinase deficiency. Indium scan, computed tomography (CT) scan, and myelography were helpful in diagnosis. Surgery and radiotherapy were followed by complete recovery during a follow-up period of 12 months.