Fluoro-Jade B evidence of induced ischemic tolerance in the rat spinal cord ischemia: physiological, neurological and histopathological consequences

Fluoro-Jade B, a marker of degenerating neurons, was used to label histopathological changes in the rat spinal cord after transient ischemia and ischemic preconditioning (IPC). To characterize postischemic neurodegenerations and consequent neurological changes, a particular attention was paid to the standardization of ischemic conditions in animals of both groups. 1. The control ischemic rats were submitted to a reversible occlusion of descending aorta by insertion and subsequent inflation of a 2F Fogarty catheter for 12 min. 2. In the IPC rats, an episode of short 3 min occlusion and 30 min reperfusion preceded the 12 min ischemia. Postischemic motor function testing (ambulation and stepping) was provided repeatedly for evaluation of neurological status 2 h and 24 h after surgery and at the end of postischemic survival, i.e. after 48 h. Fluoro-Jade B staining was used to demonstrate degenerated neurons. In the control rats, neurological consequences of histopathological changes in lumbosacral spinal cord, manifested as paraplegia, were present after 12 min ischemia. Thus, numbers of degenerated Fluoro-Jade B positive cells were visible in gray matter of the most injured L(4)-S(2) spinal cord segments. Slight motor function impairment, consequential from significant decreasing in Fluoro-Jade B-positivity in the L(4)-S(2) spinal cord segments of the IPC rats, was considered the pathomorpfological evidence that IPC induces spinal cord tolerance to ischemia. Our results are consistent with the previously published silver impregnation method for histopathological demonstration of ischemic degeneration.     

Phosphorylation of Extracellular Signal-Regulated Kinases 1/2 Predominantly Enhanced in the Microglia of the Rat Spinal Cord Following Lipopolysaccharide Injection

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord to systemic inflammation by using Western blotting and immunohistochemical techniques in a rat model intraperitoneally injected with 10 mg/kg of lipopolysaccharide (LPS). The results showed that there was a considerable amount of phosphorylated ERK 1/2 protein in the spinal cord of inflamed animals killed under pentobarbital anesthesia. The result of Western blotting showed that the phosphorylation level of ERK 1/2 in the spinal cord was increased at one hour; then 12 and 24 h after LPS injection the level decreased, while the total ERK 1/2 level seemed unchanged. The phosphorylated ERK 1/2 dominantly existed in the microglia cells of the gray matter of spinal cord, as demonstrated with double immunofluorescent staining 1 h after LPS injection. Collectively, the present results suggest that ERK signal pathway involve the cellular activation in the spinal cord following systemic inflammation, with ERK mainly in microglia. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after LPS injection implicates that ERK signaling pathway involves intracellular activity of microglia responding to the inflammation. Dan Zhou and Min Fei contributed equally to this work.     

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats.

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.     

Stress-induced phosphoprotein 1 restrains spinal cord ischaemia-reperfusion injury by modulating NF-κB signalling

Spinal cord injury (SCI), a major cause of disability, causes high global disease and economic burdens. Stress-induced phosphoprotein 1 (STIP1) has been identified to be involved in spinal cord ischaemia-reperfusion injury (SCII); however, the effect of STIP1 on SCII remains unclear until now. This study aimed to examine the role of STIP1 in SCII and unravel the possible mechanisms. Western blotting and immunohistochemical staining showed that STIP1 expression rapidly increased and then decreased in rat spinal cord following SCII treatment. Neurological function scoring, HE staining, immunohistochemical staining and Western blotting revealed that STIP1 overexpression alleviated SCII-induced motor dysfunction of hind limbs, neuronal loss and inflammation in spinal cord, and inhibited activity of nuclear factor kappa B (NF-κB) signalling in rats. Immunoprecipitation identified that STIP1 was co-located with Iba-1. In addition, STIP1 was found to ameliorate oxygen and glucose deprivation (OGD)-induced inflammation and activation of NF-κB signalling in mouse microglia BV2 cells, and STIP1 resulted in decrease of heat shock protein family A member 8 (HSPA8), increase of IκBβ expression and reduced binding of IκBβ to HSPA8 in BV2 cells. The results of the present study demonstrate that STIP1 alleviates ischaemia/reperfusion-induced neuronal injury and inflammation in rat spinal cord and mouse microglial cells by deactivating NF-κB signalling. These findings may provide novel insights for the clinical diagnosis and treatment of SCI.     

Pain modality and spinal glia expression by streptozotocin induced diabetic peripheral neuropathy in rats

Pain symptoms are a common complication of diabetic peripheral neuropathy or an inflammatory condition. In the most experiments, only one or two evident pain modalities are observed at diabetic peripheral neuropathy according to experimental conditions. Following diabetic peripheral neuropathy or inflammation, spinal glial activation may be considered as an important mediator in the development of pain. For this reason, the present study was aimed to address the induction of pain modalities and spinal glial expression after streptozotocin injection as compared with that of zymosan inflammation in the rat. Evaluation of pain behavior by either thermal or mechanical stimuli was performed at 3 weeks or 5 hours after either intravenous streptozotocin or zymosan. Degrees of pain were divided into 4 groups: severe, moderate, mild, and non-pain induction. On the mechanical allodynia test, zymosan evoked predominantly a severe type of pain, whereas streptozotocin induced a weak degree of pain (severe+moderate: 57.1%). Although zymosan did not evoke cold allodynia, streptozotocin evoked stronger pain behavior, compared with zymosan (severe+moderate: 50.0%). On the other hand, the high incidence of thermal hyperalgesia (severe+moderate: 90.0%) and mechanical hyperalgesia (severe+moderate: 85.7%) by streptozotocin was observed, as similar to that of zymosan. In the spinal cord, the increase of microglia and astrocyte was evident by streptozotocin, only microglia was activated by zymosan. Therefore, it is recommended that the selection of mechanical and thermal hyperalgesia is suitable for the evaluation of streptozotocin induced diabetic peripheral neuropathy. Moreover, spinal glial activation may be considered an important factor.     

Expression of G-protein-coupled receptor kinase 6 (GRK6) after acute spinal cord injury in adult rat

Spinal cord injury frequently results in permanent loss of neurological function. It includes many complex molecular and biochemical mechanisms. G-protein-coupled receptor kinase 6 (GRK6) is an intracellular kinase that regulates the sensitivity of certain G-protein-coupled receptors. Some studies reported GRK2 and GRK5 modulate the NFκB pathway in macrophages. Additionally, GRK2 is referred to as regulating activation of spinal cord microglia and GRK6 expression is significantly elevated in most brain regions in the MPTP-lesioned parkinsonian monkeys. However, the expression and function of GRK6 in nervous system lesion and repair are not well understood. In this study, we performed an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed the expression of GRK6 was upregulated significantly at protein level in spinal cord after SCI. Immunohistochemistry and immunofluorescence revealed wide expression of GRK6 in the normal spinal cord. After injury, GRK6 expression was increased predominantly in microglia, which expressed F4/80 (marker of macrophages and activated microglia) strongly. To understand whether GRK6 played a role in microglia activation, we applied lipopolysaccharide (LPS) to induce microglia activation in vitro. Western blot analysis demonstrated up-regulation in GRK6 protein expression after LPS stimulation was time- and dose-dependent and that up-regulation in F4/80 expression was concomitant with GRK6. These data suggested that GRK6 might be involved in the pathophysiology of SCI.     

Two faces of chondroitin sulfate proteoglycan in spinal cord repair: a role in microglia/macrophage activation

Background

Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study.

Methods and Findings

We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-α) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells.

Conclusions

Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair.     

Role of macrophages during Theiler's virus infection.

Theiler's virus, a murine picornavirus, causes a persistent infection of the central nervous system with chronic inflammation and primary demyelination. We examined the nature of infected cells at different times postinoculation (p.i.) with a combined immunocytochemistry-in situ hybridization assay. The virus was found in the gray matter of the brain, mostly in neurons, during the first week p.i. During the following weeks, the virus was present in the spinal cord, first in the gray and white matter, then exclusively in the white matter. Approximately 10% of infected cells were astrocytes at any time during the study. Infected oligodendrocytes were first noticed on day 14 p.i. and amounted to approximately 6% of infected cells. The number of infected macrophages increased with time and reached a plateau by day 21 p.i., when at least 45% of infected cells were macrophages. The role of blood-borne macrophages during infection was studied by depleting them with mannosylated liposomes containing dichloromethylene diphosphonate. The virus did not persist in the majority of the mice treated with liposomes. These mice showed only minimal mononuclear cell infiltration and no demyelination.     

Ependyma as a Possible Morphological Basis of Ischemic Preconditioning Tolerance in Rat Spinal Cord Ischemia Model: Nestin and Fluoro-Jade B Observations

1. To test our hypothesis that a transient nonlethal ischemic insult benefits the lumbosacral spinal cord ischemic injury, nestin, the marker of proliferating cells, and Fluoro-Jade B, the marker of degenerating cells, were used in rats. Morphological outcome was evaluated after 12-min ischemia versus 12-min ischemia preconditioned by 3-min ischemic period and 30-min recirculation (IPC), in each group followed by 2, 3, and 4 days of posttreatment survival. 2. Twelve-minute ischemia, inducing nestin-positivity in ependyma and reactive astrocytes at the L(1-3) spinal cord segments, shows this region as the viable region of spinal cord in all postischemic survival periods. On the other hand, abundance of Fluoro-Jade B-positive cells, distributed throughout the dorsal horn and intermediate zone of L4-S2 segments, points out the most injured spinal cord region by ischemia. 3. After the same ischemic insult in IPC rats only a few nestin-positive ependymal cell and reactive astrocytes appeared beside the nestin-positive vessels in the lower lumbar and sacral spinal cord segments of all survival periods. The appearance of nestin-positive cells in the spinal cord segments, which "should have been affected" by ischemia indicates protection of this region by the IPC treatment. 4. The number and density evaluation of Fluoro-Jade B fluorescent cells of L4-S2 segments after ischemia and IPC confirmed that degenerating cells were significantly reduced in the IPC rats in all survival periods. 5. Our results showing the immunohistochemical response of epemdyma, committed to the presence of viable tissue, indicate that the ependymal cells may contribute to the ischemic resistance in the IPC rats.     

Acute traumatic spinal cord injury induces glial activation in the cynomolgus macaque (Macaca fascicularis)

Background Traumatic spinal cord injury leads to direct myelin and axonal damage and leads to the recruitment of inflammatory cells to site of injury. Although rodent models have provided the greatest insight into the genesis of traumatic spinal cord injury (TSCI), recent studies have attempted to develop an appropriate non‐human primate model. Methods We explored TSCI in a cynomolgus macaque model using a balloon catheter to mimic external trauma to further evaluate the underlying mechanisms of acute TSCI. Results Following 1 hour of spinal cord trauma, there were focal areas of hemorrhage and necrosis at the site of trauma. Additionally, there was a marked increased expression of macrophage‐related protein 8, MMP9, IBA‐1, and inducible nitric oxide synthase in macrophages and microglia at the site of injury. Conclusions This data indicate that acute TSCI in the cynomolgus macaque is an appropriate model and that the earliest immunohistochemical changes noted are within macrophage and microglia populations.     

Prolonged Gray Matter Disease without Demyelination Caused by Theiler's Murine Encephalomyelitis Virus with a Mutation in VP2 Puff B

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups based on neurovirulence. During the acute phase, DA virus infects cells in the gray matter of the central nervous system (CNS). Throughout the chronic phase, DA virus infects glial cells in the white matter, causing demyelinating disease. Although GDVII virus also infects neurons in the gray matter, infected mice developed a severe polioencephalomyelitis, and no virus is detected in the white matter or other areas in the CNS in rare survivors. Several sequence differences between the two viruses are located in VP2 puff B and VP1 loop II, which are located near each other, close to the proposed receptor binding site. We constructed a DA virus mutant, DApBL2M, which has the VP1 loop II of GDVII virus and a mutation at position 171 in VP2 puff B. While DApBL2M virus replicated less efficiently than DA virus during the acute phase, DApBL2M-induced acute polioencephalitis was comparable to that in DA virus infection. Interestingly, during the chronic phase, DApBL2M caused prolonged gray matter disease in the brain without white matter involvement in the spinal cord. This is opposite what is observed during wild-type DA virus infection. Our study is the first to demonstrate that conformational differences via interaction of VP2 puff B and VP1 loop II between GDVII and DA viruses can play an important role in making the transition of infection from the gray matter in the brain to the spinal cord white matter during TMEV infection.     

Upregulation of Vascular Endothelial Growth Factor Receptor-3 in the Spinal Cord of Lewis Rats with Experimental Autoimmune Encephalomyelitis

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor–3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.     

White matter injury following systemic endotoxemia or asphyxia in the fetal sheep

White matter injury is the most frequently observed brain lesion in preterm infants. The etiology remains unclear, however, both cerebral hypoperfusion and intrauterine infections have been suggested as risk factors. We compared the neuropathological outcome, including the effect on oligodendrocytes, astrocytes, and microglia, following either systemic asphyxia or endotoxemia in fetal sheep at midgestation. Fetal sheep were subjected to either 25 minutes of umbilical cord occlusion or systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (LPS O111:B4, 100 ng/kg, IV). Periventricular white matter lesions were observed in 2 of 6 asphyxiated fetuses, whereas the remaining animals showed diffuse injury throughout the subcortical white matter and neuronal necrosis in subcortical regions, including the striatum and hippocampus. LPS-treatment resulted in focal inflammatory infiltrates and cystic lesions in periventricular white matter in 2 of 5 animals, but with no neuron specific injury. Both experimental paradigms resulted in microglia activation in the white matter, damaged astrocytes, and loss of oligodendrocytes. These results show that the white matter at midgestation is sensitive to injury following both systemic asphyxia and endotoxemia. Asphyxia induced lesions in both white and subcortical grey matter in association with microglia activation, and endotoxemia resulted in selective white matter damage and inflammation.     

Role of VP2 amino acid 141 in tropism of Theiler's virus within the central nervous system.

Following intracranial inoculation, Theiler's virus causes either an acute encephalitis (strain GDVII) or a chronic demyelinating disease (strain DA). The DA strain sequentially infects the grey matter of the brain, the grey matter of the spinal cord, and, finally, the white matter of the spinal cord, where it persists in glial cells and causes demyelinating lesions. Analysis of the phenotype of recombinant viruses has shown that the viral capsid contains determinants for persistence and demyelination. Our previous studies showed that a Lys at position 141 of the VP2 capsid protein (VP2-141) could render a chimeric virus persistent. We also reported that another recombinant virus, virus R5, migrated from the grey matter of the brain to that of the spinal cord inefficiently and was unable to infect the white matter of the spinal cord. In this article, we report that introducing a Lys at position VP2-141 in virus R5 increases its ability to infect the white matter of the spinal cord. Our results indicate that this amino acid is important for the spread of the virus within the central nervous system.     

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.     

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.     

Toll-like Receptor 2 Mediates Peripheral Nerve Injury-induced NADPH Oxidase 2 Expression in Spinal Cord Microglia

We have previously reported that NADPH oxidase 2 (Nox2) is up-regulated in spinal cord microglia after spinal nerve injury, demonstrating that it is critical for microglia activation and subsequent pain hypersensitivity. However, the mechanisms and molecules involved in Nox2 induction have not been elucidated. Previous studies have shown that Toll-like receptors (TLRs) are involved in nerve injury-induced spinal cord microglia activation. In this study, we investigated the role of TLR in Nox2 expression in spinal cord microglia after peripheral nerve injury. Studies using TLR knock-out mice have shown that nerve injury-induced microglial Nox2 up-regulation is abrogated in TLR2 but not in TLR3 or -4 knock-out mice. Intrathecal injection of lipoteichoic acid, a TLR2 agonist, induced Nox2 expression in spinal cord microglia both at the mRNA and protein levels. Similarly, lipoteichoic acid stimulation induced Nox2 expression and reactive oxygen species production in primary spinal cord glial cells in vitro. Studies on intracellular signaling pathways indicate that NF-κB and p38 MAP kinase activation is required for TLR2-induced Nox2 expression in glial cells. Conclusively, our data show that TLR2 mediates nerve injury-induced Nox2 gene expression in spinal cord microglia via NF-κB and p38 activation and thereby may contribute to spinal cord microglia activation.     

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates

Microglia are the resident macrophages in the central nervous system. In the spinal cord dorsal horn, microglia stay in resting condition during physiological sensory processing, and are activated under pathological conditions such as peripheral nerve injury. In cases such as this, the nearby resting microglia increase their motility and accumulate at the site of injury. However, direct evidence to support that nerve activity can enhance the motility of microglia has not yet to be reported. In this study we investigated whether the activation of spinal microglia under in vivo nerve injury may be mimicked by neuronal activity in the spinal cord slice preparation. We found that local application of spinal excitatory neurotransmitters, such as glutamate and substance P did not cause any change in the motility of microglial cells in the spinal cord dorsal horn. The motility of microglial cells is unlikely modulated by other transmitters, neuromodulators and chemokines, because similar applications such as GABA, serotonin, noradrenaline, carbachol, fractalkine or interleukin did not produce any obvious effect. Furthermore, low or high frequency stimulation of spinal dorsal root fibers at noxious intensities failed to cause any enhanced extension or retraction of the microglia processes. By contrast, focal application of ATP triggered rapid and robust activation of microglial cells in the spinal dorsal horn. Our results provide the first evidence that the activation of microglia in the spinal cord after nerve injury is unlikely due solely to neuronal activity, non-neuronal factors are likely responsible for the activation of nerve injury-related microglial cells in the spinal dorsal horn.