Growth associated protein 43 and neurofilament immunolabeling in the transected lumbar spinal cord of lizard indicates limited axonal regeneration

Previous cytological studies on the transected lumbar spinal cord of lizards have shown the presence of differentiating glial cells,few neurons and axons in the bridge region between the proximal and distal stumps of the spinal cord in some cases.A limited number of axons(20-50)can cross the bridge and re-connect the caudal stump of the spinal cord with small neurons located in the rostral stump of the spinal cord.This axonal regeneration appears to be related to the recovery of hind-limb movements after initial paralysis.The present study extends previous studies and shows that after transection of the lumbar spinal cord in lizards,a glial-connective tissue bridge that reconnects the rostral and caudal stumps of the interrupted spinal cord is formed at 11-34 days post-injury.Following an initial paralysis some recovery of hindlimb movements occurs within 1-3 months post-injury.Immunohistochemical and ultrastructural analysis for a growth associated protein 43(GAP-43)of 48-50 k Da shows that sparse GAP-43 positive axons are present in the proximal stump of the spinal cord but their number decreased in the bridge at 11-34 days post-transection.Few immunolabeled axons with a neurofilament protein of 200-220 k Da were seen in the bridge at 11-22 days post-transection but their number increased at 34 days and 3 months post-amputation in lizards that have recovered some hindlimb movements.Numerous neurons in the rostral and caudal stumps of the spinal cord were also labeled for GAP43,a cytoplasmic protein that is trans-located into their axonal growth cones.This indicates that GAP-43 biosynthesis is related to axonal regeneration and sprouting from neurons that were damaged by the transection.Taken together,previous studies that utilized tract-tracing technique to label the present observations confirm that a limited axonal re-connection of the transected spinal cord occurs 1-3 months post-injury in lizards.The few regenerating-sprouting axons within the bridge reconnect the caudal with the rostral stumps of the spinal cord,and likely contribute to activate the neural circuits that sustain the limited but important recovery of hind-limb movements after initial paralysis.The surgical procedures utilized in the study followed the regulations on animal care and experimental procedures under the Italian Guidelines(art.5,DL 116/92).     

Synaptogenesis in the cervical cord of the human embryo: sequence of synapse formation in a spinal reflex pathway.

Synaptogenesis in the cervical cord was studied by light and electron microscopy in human embryos ranging from four to seven weeks of ovulation age. The stage of embryonic development was estimated on the basis of external morphology of embryos and histology of the eye ball with reference to Streeter's horizon. No synapses were found in the cervical cord of the embryo at Streeter's horizon XIV (8 mm; estimated ovulation age, 28-30 days). A small number of axodendritic synapses appear in the motor neuropil of the cervical cord at Streeter's horizon XVII (14 mm; estimated ovulation age, 34-36 days). Since no primary afferents are demonstrated to reach the motor neuropil at this stage (the premotile period), these synapses are considered to be formed between interneurons and motor neurons. On the other hand, the formation of synapses outside the motor neuropil of the cervical cord was recognized at Streeter's horizon XX (22 mm; estimated ovulation age, 40-42 days), which corresponded to the period of onset of the precocious reflex, but not by horizon XIX (18 mm; estimated ovulation age, 38-40 days). The first axosomatic synapses were found in the motor neuropil at Streeter's horizon XVII (estimated ovulation age, 34-36 days). The present study suggests that the formation of synapses between interneurons and dendrites of spinal motor neurons precedes that of synapses between interneurons and collaterals of primary afferents. This sequence of synaptogenesis is in agreement with that reported in earlier studies with silver stain methods.     

Spinal cord injury is accompanied by chronic progressive demyelination

Preceding the development of therapeutic strategies for spinal cord injury is an identification of those pathological processes that might serve as therapeutic targets. Although demyelination has been documented as a secondary degenerative component of spinal cord injury in several species including humans, the extent of demyelination and its functional consequence remain unknown. In this report, we document the extent of demyelination and remyelination up to 450 days following contusive spinal cord injury in adult rats. The overall number of demyelinated axons peaked at 1 day post injury, declined by 7-14 days post injury, and then progressively increased up to 450 days post injury. Oligodendrocyte and Schwann cell remyelinated axons appeared by 14 days post injury. Although remyelinated axons were present from 14 to 450 days post injury, remyelination was incomplete, as indicated by the presence of demyelinated axons at every time point examined. These studies demonstrate for the first time that spinal cord injury is accompanied by chronic progressive demyelination, and they substantiate demyelination as a target for therapeutic intervention.     

Neuropathologic effects of intrathecal water.

In attempting to control the pain of disseminated carcinoma, a variety of agents has been injected intrathecally. This report deals with the neuropathologic changes in spinal roots and spinal cord of four patients into whose lumbar sacs 100 to 285 ml of distilled water was infused. Survival following the procedure ranged from 9 to 50 days. In most severely affected case (285 ml instilled 50 days prior to death) the abnormalities were: (1) severe breakdown of axons and myelin sheaths of the lower cauda equina and to a lesser extent of other roots at levels as high as the eighth cervical segment, (2) severe central chromatolysis of anterior horn cells in lumbo-sacral spinal cord with milder degrees of change in the thoracic and lower cervical spinal cord, and (3) striking vacuolar change and axonal swelling, primarily in the lateral columns ascending as high as the lower cervical spinal cord. A second case (140 ml instilled 24 days prior to death) showed similar but milder changes. The remaining cases (100 ml instilled 34 days prior to death: 100 and 200 ml instilled 27 and 9 days prior to death) showed no changes referable to intrathecal water. Infusion of distilled water intrathecally is capable of producing distinctive lesions of spinal root and cord.     

Activation of p38 mitogen-activated protein kinase in spinal microglia mediates morphine antinociceptive tolerance

Compelling evidence has suggested that spinal glial cells were activated by chronic morphine treatment and involved in the development of morphine tolerance. However, the mechanisms of glial activation were still largely unknown in morphine tolerance. In present study, we investigated the role of p38 mitogen-activated protein kinase (p38 MAPK) in the spinal cord in the development of chronic morphine antinociceptive tolerance. We found that intrathecal administration of morphine (15 microg) daily for 7 consecutive days significantly induced an increase in number of phospho-p38 (p-p38) immunoreactive cells in the spinal cord compared with chronic saline or acute morphine treated rats. Double immunofluorescence staining revealed that p-p38 immunoreactivity was exclusively restricted in the activated spinal microglia, not in astrocytes or neurons. Repeated intrathecal administration of 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (10 microg or 2 microg), a specific p38 inhibitor, 30 min before each morphine injection for 7 consecutive days significantly attenuated tolerance to morphine analgesia assessed by tail flick test. However, a single intrathecal administration of SB203580 (10 microg) did not antagonize the established tolerance to morphine analgesia. Taken together, these findings suggested that p38 MAPK activation in the spinal microglia was involved in the development of morphine antinociceptive tolerance. Inhibition of p38 MAPK by SB203580 in the spinal cord attenuated but not reversed the tolerance to morphine analgesia. The present study provides the first evidence that p38 activation in spinal microglia played an important role in the development of tolerance to morphine analgesia.     

Development of transferrin-positive oligodendrocytes in the rat central nervous system

Transferrin is the second most abundant plasma protein and functions to transport iron. It is an essential constituent in culture media for virtually all cells. In a recent study, we reported that transferrin (Tf) is specifically located in oligodendrocytes in the rat nervous system. This investigation examines immunohistochemically the development of Tf in the cerebral cortex, corpus striatum, and spinal cord. Tf is first seen in oligodendrocytes in the spinal cord white matter at 5 days of age. The immunoreactivity is confined to the white matter in the periphery of the spinal cord between 5 and 8 days of age. By 10-12 days of age, the number of immunoreactive oligodendrocytes in the spinal cord white matter increases considerably, corresponding to the onset of myelination. Tf-positive oligodendrocytes are first found in the gray matter at 15 days of age. By 30 days of age, the number and distribution of Tf-positive oligodendrocytes in both the brain and spinal cord have reached the adult pattern. The results of this study demonstrate a spatial and temporal association between Tf development and myelinogenesis. This suggests that part of the process of differentiation of oligodendrocytes includes the accumulation of Tf, perhaps in order to support the metabolic demands associated with the production and maintenance of myelin.     

Developmental patterns of neurite outgrowth from chick embryo spinal cord and retinal neurons on laminin substrates

It has been previously found that neurite outgrowth on collagen substrates decreases with increasing gestational age of chick embryo spinal cord and retinal neurons in tissue culture. In the current study, laminin, polylysine and collagen were compared in their efficacy in promoting neurite extension from chick embryo spinal cord neurons aged 6-16 days or retinal neurons aged 8-16 days in ovo. The percentage of neurons with neurites and the length of the neurites were determined at 1 and 3 days in culture. There was a significant increase in neuritogenesis by laminin and polylysine compared to collagen for both spinal cord and retinal neurons. Further, in spinal cord cultures grown on a laminin substrate, there was no decline in neurite outgrowth with increasing developmental age of the neurons as was seen on collagen and polylysine. Neurite length measurements also demonstrated a significant stimulation of neuritogenesis for spinal cord, but not retinal, neurons by laminin compared to polylysine or collagen in 1-day cultures. The results demonstrate tissue-specific differences in the developmental patterns of neurite outgrowth. Retinal neurons appear to have intrinsic changes in their ability to respond to extracellular promoting factors or substrates, while spinal cord neurite outgrowth can be regulated by these extrinsic factors.     

Development of serotonin immunoreactivity in the rat spinal cord and its plasticity after neonatal spinal cord lesions

The postnatal maturation of spinal pathways may account for the gradual time course of postnatal development of behavior and also account for the greater anatomical reorganization which often follows damage to the developing CNS compared to the mature CNS. The purpose of the current study was to examine (1) the prenatal and postnatal development of the descending serotonergic (5-HT) projection to the spinal cord and (2) the effects of a neonatal spinal cord lesion on this development. In addition, we wished to determine (3) whether transplants of fetal spinal cord tissue placed into the neonatal lesion site alter the plasticity of the 5-HT projection to the cord. Peroxidase-antiperoxidase immunocytochemical techniques were used. At embryonic day 14 (E14), no 5-HT immunoreactive fibers could be identified at any spinal cord level. By E18 the first axons were identified in the white matter only at all spinal cord levels. At birth, 5-HT immunoreactive fibers were present both in the white matter and in the gray matter at all cord levels. The projection within the gray matter was diffuse and considerably less dense than in the adult. The postnatal maturation of the 5-HT projection within the gray matter of the spinal cord followed rostral to caudal and ventral to dorsal gradients. During the first weeks postnatal, the 5-HT immunoreactivity within the cord increased to attain an adult pattern and density by 14 days in the cervical cord and 21 days in the thoracic and lumbar cord. The effect of a spinal cord hemisection at birth on the anatomical reorganization of the descending serotonergic innervation of the cord was compared with the effect of the same lesion in the adult. In the adult animal, mid-thoracic hemisection decreased the 5-HT content of the ventral horn of the lumbar spinal cord caudal and ipsilateral to the lesion to 8% of that on the intact side. When this same lesion was made in the newborn animal, the innervation was 43% of that on the intact side. When a transplant of fetal spinal cord tissue was inserted into the lesion site in the newborn animals, there was even greater 5-HT innervation caudal to the lesion, 83% of that on the intact side. These results indicate that there is considerable postnatal development and plasticity of the descending serotonergic projection to the spinal cord, and this plasticity is enhanced by the presence of a spinal cord transplant at the site of the lesion.     

Autoradiographic distribution and alterations of kinin B(2) receptors in the brain and spinal cord of streptozotocin-diabetic rats

This study investigates whether bradykinin (BK) B(2) receptor binding sites are increased in the brain and thoracic spinal cord of streptozotocin (STZ)-diabetic rats at 2, 7, and 21 days posttreatment by in vitro autoradiography with the radioligand [(125)I]HPP-Hoe 140. In control and diabetic rats, specific binding sites for B(2) receptors were detected in the brain and in various laminae of the spinal cord, predominantly in superficial laminae (K(d)=34 pM). In diabetic rats, B(2) receptor densities were significantly increased in lamina l of the dorsal horn (+35% at 7 and 21 days), spinal trigeminal nucleus (+70% at 7 and 21 days) and nucleus tractus solitarius (+100% at 2 and 7 days). B(2) receptor analogues D-Arg[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-BK (Hoe 140), 3-(4 hydroxyphenyl)propionyl-Hoe 140 (HPP-Hoe 140), LF16-0687 mesylate ((2-Pyrrolidinecarboxamide, N-[3-[[4-aminoiminomethyl)benzoyl]amino]propyl]-1-[[2,4-dichoro-3-[[(2,4-dimethyl-8-quinolinyl)oxy]methyl]phenyl]sulfonyl]-(2S)-(9Cl)), and BK decreased binding of [(125)I]-HPP-Hoe 140 in the spinal dorsal horn, with K(i) values of 0.5, 1.5, 3.2, and 3.7 nM, respectively. These values were not significantly different in diabetic rats at 7 days (0.5 (Hoe 140), 0.7 (HPP-Hoe 140), 1.2 (BK), and 1.7 (LF16-0687) nM). While des-Arg(10)-Hoe 140 was three orders of magnitude less potent than Hoe 140, B(1) receptor agonist (des-Arg(9)-BK) and antagonist (AcLys[D-betaNal(7),Ile(8)]des-Arg(9)-BK, R-715) did not affect [(125)I]-HPP-Hoe 140 binding at 1 microM concentration. Data suggest a very discrete and temporal increase of B(2) receptor density (without affinity changes) in the spinal cord and hindbrain of STZ-diabetic rats. This contrasts with the early induction and over-expression of B(1) receptors reported in the brain and spinal cord of STZ-diabetic rats.     

Neuronal Apoptosis and Necrosis Following Spinal Cord Ischemia in the Rat

We examined the characteristics of neuronal death induced by ischemia in the spinal cord. Spinal cord ischemia was induced in Long–Evans rats by occlusion of the descending aorta with a 2F Fogarty catheter for 20 min (model 1) or more limited aortic occlusion (15 min) coupled with blood volume reduction (model 2); rats were sacrificed 6 h–7 days later. The animals developed variable paraparesis in model 1 and reliable paraplegia in model 2. The extent of histopathological spinal cord damage, being maximal in the lumbar cord, correlated well with the severity of paraparesis. Two distinct types of spinal cord neuronal death were observed, consistent with necrosis and apoptosis. Neuronal necrosis was seen in gray matter laminae 3–7, characterized by the rapid (6 h) onset of eosinophilia on hematoxylin/eosin-stained sections, and gradual (1–7 days) development of eosinophilic ghosting. Although TUNEL positivity was present, disintegration of membranes and cytoplasmic organelles was seen under electron microscopy. Neuronal apoptosis was seen after 1–2 days in dorsal horn laminae 1–3, characterized by both TUNEL positivity and electron microscopic appearance of nuclear chromatin aggregation and the formation of apoptotic bodies. DNA extracted from the ischemic lumbar cord showed internucleosomal fragmentation (laddering) on gel electrophoresis. These data suggest that distinct spinal cord neuronal populations may undergo necrosis and apoptosis following transient ischemic insults.     

A Quantitative Spatial Analysis of the Blood–Spinal Cord Barrier: II. Permeability after Intraspinal Fetal Transplantation

In previous experiments we utilized quantitative autoradiography to temporally describe vascular permeability of a radiolabeled vascular tracer following spinal contusion injury in the rat. In the present report we compare these findings with permeability assessments following fetal grafting in the contused rat spinal cord. At 10 days postinjury, Embryonic Day 14 spinal tissue was grafted into the lesioned spinal cord of Sprague–Dawley rats. At 7, 14, and 28 days postgrafting the α-aminoisobutyric acid (AIB) technique was used to assess blood-to-tissue transfer rates in graft and host tissue over several segments of the injured spinal cord. Regional changes in permeability were assessed using four distinct image analysis techniques. Using these methods, we have previously shown that contusion injury alone results in a chronic relapse in vascular permeability. The present data indicate that fetal transplants at 7 days postgrafting have AIB transfer rates that are significantly above uninjured control levels and are similar in magnitude to neighboring host spinal tissue. In addition, permeability in 14- and 28-day intraspinal grafts decreased relative to that of the 7-day transplant group, but remained significantly elevated at and rostral to the injury epicenter. Alternately, graft and host tissue in regions caudal to the injury epicenter (e.g., T₁₀–L₂) acquired a functional barrier to AIB as early as 14 days posttransplantation. These experiments suggest that graft development occurs in a different manner or at a different rate in segments of the injured spinal cord rostral and caudal to the injury site. Additionally, it appears that vascular permeability of the injured spinal cord can be influenced by the process of intraspinal transplantation.     

Close spatial-temporal relationship between islet-1-expressing cells and growing primary afferent axons in the dorsal spinal cord of chick embryo

The relationship between the appearance of Islet-1-expressing cells and the longitudinal growth of primary afferent axons (PAAs) in the dorsal spinal cord of chick embryos was examined. Islet-1-expressing cells first appeared in the dorsal spinal cord at embryonic days (E) 3-3.5. These immunoreactive cells were aligned in a longitudinal column in close proximity to longitudinally elongating PAAs in the presumptive dorsal funiculus. By E8, when many PAAs invade the spinal gray matter, Islet-1-expressing cells had disappeared in the dorsal spinal cord. Following the dorsoventral rotation of the spinal cord in ovo before the invasion of PAAs, a close topographical relationship between Islet-1-expressing cells and PAAs was maintained. These results suggest that Islet-1-expressing cells may play a role in the longitudinal growth of PAAs in the dorsal funiculus.     

Muscle-derived factors reverse the cholinotoxic effects of ethanol during early neuroembryogenesis in the chick embryo.

The interaction between muscle-derived factors and ethanol on cholinergic neuronal expression was studied in the chick embryo during early neuroembryogenesis using choline acetyltransferase (ChAT) as cholinergic neuronal marker. Ethanol (10 mg/50 microliters) and limb muscle extract (130 micrograms protein/50 microliters) (LME) were administered in ovo either alone or concomitantly at embryonic days 1-3 (E1-E3); or ethanol was given E1-E3 and followed by LME at E4-E7. All groups were sacrificed at embryonic day 8 (E8) and ChAT activity was assayed in homogenates of whole brain and of spinal cord. As previously reported, ethanol at E1-3 produced a 30% decrease in brain ChAT activity and 35% in spinal cord. Concomitant administration of ethanol and LME at embryonic days E1-E3 eliminated the decrease in choline acetyltransferase activity produced by ethanol in the brain, but not in the spinal cord. On the other hand, administration of LME at embryonic days E4-E7 to embryos pretreated with ethanol at embryonic days E1-E3, raised ChAT activity to control level in the spinal cord, but only partially restored ChAT activity in the brain. In view of the alleged neurotrophic effects of muscle-derived factors on neuronal survival and neuronal growth, we interpret these findings to suggest that LME in addition to its ability to decrease natural neuronal death, may prevent death resulting from neurotoxicity.     

Cytomegalovirus infection of the developing brain alters catecholamine and indoleamine metabolism

Tissue concentrations of noradrenaline (NA), serotonin (5-HT), dopamine (DA) and selected metabolites were measured in the spinal cord, cerebellum, cerebral cortex and caudate-putamen of developing mice following intraventricular inoculation with murine cytomegalovirus (MCMV) on postnatal day 10. MCMV-infected animals exhibited transient signs of neurological impairment, including apparent hypertonicity of hindlimb extensors and abnormal gait, beginning on days 14-16 and continuing for 3-5 days. At the onset of neurological impairment, tissue concentrations of NA were significantly reduced in the spinal cord (20%), cerebellum (32%) and cerebral cortex (40%) of infected animals. Levels of 5-HT were significantly increased in the caudate-putamen (50%), while 5-hydroxyindoleacetic acid (5-HIAA) was increased in both the spinal cord (94%) and caudate-putamen (65%). The ratio of 5-HIAA/5-HT, which is frequently used as an estimate of turnover of 5-HT, was significantly increased in the spinal cord (90%) at the onset of neurological impairment. In the caudate-putamen of MCMV-infected animals, there were significant increases in the tissue levels of DA (37%), homovanillic acid (HVA, 41%) and 3,4-dihydroxyphenylacetic acid (DOPAC, 34%). All neurochemical parameters were normal in the MCMV-infected animals by postnatal day 70, approximately 50 days after the resolution of neurological signs. These results indicate transient alterations in monoamine metabolism in the developing nervous system during the pathogenesis of cytomegalovirus-induced movement and postural disorders.     

Ontogenesis of adenosine receptors in the central nervous system of the rat

The ontogeny of adenosine receptors was studied in rat brain and spinal cord using the specific ligand [3H]cyclohexyladenosine [( 3H]CHA). The [3H]CHA affinity constant (Kd) and the maximum receptor binding capacity (Bmax) were analyzed at all ages and in all CNS regions studied. Throughout development the Kd of [3H]CHA binding remained relatively stable and for cortex, cerebellum, subcortex, midbrain, brainstem and spinal cord ranged from 2.2 +/- 0.2 to 5.5 +/- 0.6 nM (mean +/- S.E.M.). In contrast, the Bmax values from 1- and 90-day animals increased by as little as 2-fold in subcortical regions and by as much as 9- and 16-fold in cortex and cerebellum, respectively. The highest density of binding sites was observed in subcortical structures and the lowest in brainstem and midbrain. In cortex, a steady increase in receptor number began at day 1 and stopped at the adult level by 21 days. In cerebellum, maximum receptor proliferation began at about 14 days and continued to adulthood. Other CNS regions showed intermediate rates of receptor development. These differences may reflect both the pattern of postnatal neurogenesis in the rat CNS and the maturation of those neural elements containing adenosine receptors.     

Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury★

BACKGROUND:Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March...     

Loss of microRNA-124 expression in neurons in the peri-lesion area in mice with spinal cord injury

MicroRNA-124 (miR-124) is abundantly expressed in neurons in the mammalian central ner-vous system, and plays critical roles in the regulation of gene expression during embryonic neurogenesis and postn...     

Muscle-derived factors enhance cholinergic neuronal expression in the chick embryo--I. In ovo studies

The effects of muscle-derived factors were studied in the chick embryo in ovo, during early neuroembryogenesis. Limb muscle extract (LME) administration during embryonic period E1-E7 produced a significant increase in choline acetyltransferase activity of both spinal cord and brain in 8-day-old chick embryos. Similar treatment failed to induce significant change in the GABAergic phenotypes as assessed by the activity of the enzyme glutamic acid decarboxylase. Administration of limb muscle extract at either embryonic days 1-3 or 4-7 produced a significant increase in choline acetyltransferase activity in the brain, indicating that the critical period of limb muscle extract in the brain to be between embryonic days E1 and E7, a period of neuronal proliferation and differentiation in the brain. On the other hand, LME administration produced no effect on spinal cord choline acetyltransferase activity when given at embryonic days 1-3, whereas it produced a marked increase when given at embryonic days 4-7. Thus, the critical period of limb muscle extract effect in the spinal cord appears to be confined to embryonic days E4-E7, a period of neuronal differentiation and cell death in the spinal cord. These findings indicate that the cholinotrophic activity of muscle-derived factors is not limited to the muscle target tissues but have a general effect on cholinergic neurons in the CNS. Whether these cholinotrophic effects are mediated by a common factor or by different factors is still under investigation.     

TCR peptide therapy decreases the frequency of encephalitogenic T cells in the periphery and the central nervous system

The V beta 8 CDR2 consensus peptide, residues 44-54, is highly effective in the treatment of clinical experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To monitor immunological changes during EAE resulting from TCR peptide therapy, the frequencies of encephalitogenic and regulatory T cells were quantitated in lymph nodes, blood, and spinal cord. The frequency of T cells specific for basic protein and its major encephalitogenic epitope, residues 72-89, increased during EAE to about 1 cell per 100,000 lymph node or blood cells at the peak of clinical disease, and then declined. In contrast, the frequency of these T cells in spinal cord was highest, 50 per 100,000, prior to onset of clinical signs, and then decreased rapidly prior to spontaneous recovery. Injection of 100 micrograms of TCR V beta 8-44-54 peptide caused a decrease within 1-5 days in the frequencies of guinea pig basic-protein (GP-BP) and 72-89-reactive T cells in blood and spinal cord, and in the total number of infiltrating cells in spinal cord. In lymph nodes, 72-89-reactive T cells decreased as T cells specific for a protective epitope, residues 55-69 of GP-BP increased, suggesting epitope switching at the site of GP-BP immunization. Conversely, the frequency of T cells specific for the V beta 8-44-54 peptide increased, especially in blood and spinal cord, whereas T cell frequencies to control antigens were unchanged. These data document the critical presence of encephalitogenic T cells within the spinal cord during clinical EAE, and demonstrate that rapid and profound changes in T cell frequencies in the periphery and spinal cord are triggered by TCR peptide therapy.     

Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury★

BACKGROUND: Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE: To investigate inducible nitric oxide synthase (iNOS) and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING: The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March 2007 to March 2008. MATERIALS: A total of 120 healthy, adult Sprague Dawley rats were selected for this study. Rat models of moderate acute thoracic (T9) spinal cord injury were established using the modified Allen method. Shuxuening injection was obtained from Zhenbaodao Pharmaceutical Co., Ltd., China. Methylprednisolone was purchased from North China Pharmaceutical Co., Ltd. METHODS: All rats were equally and randomly divided into four groups. Only the spinal cord was exposed in the sham operation group rats. In the trauma group, rats were not treated with drugs following spinal cord injury. Rats in the hormone group were intraperitoneally injected with 30 mg/kg methylprednisolone following spinal cord injury. Rats in the ginkgo biloba leaf extract group were intraperitoneally infused with a 1.0 mL/kg Shuxuening injection per day. MAIN OUTCOME MEASURES: At 1 hour, as well as 1, 3, 5, 7, and 14 days after spinal cord injury, iNOS- and Bcl-2/Bax-positive cells were quantified with immunohistochemistry. Pathological changes were detected using hematoxylin-eosin staining under an optical microscope. RESULTS: Spinal cord injury in the ginkgo biloba leaf extract and hormone groups was milder compared with the trauma group. Demyelination was significantly ameliorated and the necrotic cavity was obviously reduced in the injured spinal cord of rats in the ginkgo biloba leaf extract and hormone groups at each time point. iNOS expression was increased in the injured spinal cord, and reached a peak at 5 days. The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P < 0.05-0.01). The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P < 0.05). Bcl-2 expression reached a peak at 3 days, and Bax expression reached a peak at 5 days following rat spinal cord injury. Bcl-2 expression was increased, but Bax expression was decreased in the ginkgo biloba leaf extract and hormone groups compared with the trauma group (P < 0.05-0.01). Bcl-2 expression was greater, but Bax expression was reduced in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury (P < 0.05). CONCLUSION: Ginkgo biloba leaf extract exhibits neuroprotective effects by upregulating Bcl-2 expression, downregulating Bax expression, and significantly inhibiting high expressions of iNOS in the injured spinal cord. The neuroprotective effects of ginkgo biloba leaf extract are greater compared with methylprednisolone at 1 week after spinal cord injury. Key Words: apoptosis; Bcl-2/Bax; ginkgo biloba leaf extract; inducible nitric oxide synthase; methylprednisolone; neuroprotection; spinal cord injury