The role of timing in the treatment of spinal cord injury.

Regeneration failure after primary spinal cord injury (SCI) leads to diverse clinical complications in a severity- and level of SCI-dependent manner. The cost of treating both of them (initial regeneration failure and following complications) would be prohibitive, particularly in less developed nations. The well-recognized circumstances arose from primary SCI include excitotoxicity and inflammation. SCI increases concentrations of extracellular amino acids (EAAs) in the severity-dependent manner and the maximum level of EAAs at the injury site will be reduced by distance from the injury site. Increased concentrations of EAAs and their signaling result in energy and metabolic changes and eventually neurotoxicity. Therefore EAAs play a crucial role in moving towards secondary stage of SCI. There is a close correspondence between severity of SCI and intensity of acute inflammatory response, which includes proinflammatory cytokines (IL-1ß, TNF-α, and IL-6) and immune cells (neutrophils, microglia, and mast cells). The communication between microglia and astrocytes mediate formation of astroglial scar. The scar is thought to diminish the spread of inflammation and lesion volume, and on the other side poses an obstacle to achieving axon regeneration. Moreover, mast cells exert an anti-inflammatory role in the ground of injured spinal cord by degradation of proinflammatory mediators, while mast cells-derived histamine may cause excitotoxicity. Therefore research suggests a very double-sword remark about the work of inflammatory mediators in the injured spinal cord. Myelin associated inhibitors (MAIs) are among the growing list of extrinsic inhibitors of neuroregeneration in the injured-CNS. They function via NgR-dependent mechanisms. The time for intervention by NgR antagonists must be fixed according to the expression pattern of this receptor and its dependent MAIs after SCI. Altogether, experimental studies suggest potential benefits of combating EAAs, inflammatory mediators, and MAIs during the first minutes, hours and weeks after SCI, respectively. However, acute inflammation initially induced by SCI tends to be permanent, even at several years after SCI. This supports the notion that paying attention to inflammation must persist through time. The consideration of seconds-dependent state of spinal cord after primary injury is a very therapeutic and also preventive approach against future possible complications. It is thereby possible to propose "timing", which is perfectly practicable throughout the world, as an effective campaign against the final failure of SCI.

Microglia and monocytes synergistically promote the transition from acute to chronic pain after nerve injury.

Microglia and peripheral monocytes contribute to hypersensitivity in rodent models of neuropathic pain. However, the precise respective function of microglia and peripheral monocytes has not been investigated in these models. To address this question, here we combined transgenic mice and pharmacological tools to specifically and temporally control the depletion of microglia and monocytes in a mouse model of spinal nerve transection (SNT). We found that although microglia and monocytes are required during the initiation of mechanical allodynia or thermal hyperalgesia, these cells may not be as important for the maintenance of hypersensitivity. Moreover, we demonstrated that either resident microglia or peripheral monocytes are sufficient in gating neuropathic pain after SNT. We propose that resident microglia and peripheral monocytes act synergistically to initiate hypersensitivity and promote the transition from acute to chronic pain after peripheral nerve injury.

The somatostatin receptor 4 agonist J-2156 reduces mechanosensitivity of peripheral nerve afferents and spinal neurons in an inflammatory pain model.

Somatostatin (SST) is a peptide hormone that regulates the endocrine system and affects neurotransmission via interaction with G protein-coupled SST receptors and inhibition of the release of different hormones. The aim of this study was to investigate whether the analgesic properties of the selective SSTR4 agonist J-2156 are mediated via peripheral and/or spinal receptors. Effect on mechanical hyperalgesia in the Complete Freund׳s Adjuvant (CFA) model was measured after intraperitoneal application of J-2156. Electrophysiological neuronal recordings were conducted 24 h after injection of CFA or vehicle into the paw of Wistar rats. Mechanosensitivity of peripheral afferents of the saphenous nerve as well as of spinal wide dynamic range (WDR) and nociceptive-specific (NS) neurons were measured after systemic or spinal application of J-2156. In CFA animals J-2156 dose dependently reduced hyperalgesia in behavioral studies. The minimal effective dose was 0.1 mg/kg. Mechanosensitivity of peripheral afferents and spinal neurons was significantly reduced by J-2156. NS neurons were dose dependently inhibited by J-2156 while in WDR neurons only the highest concentration of 100 µM had an effect. In sham controls, J-2156 had no effect on neuronal activity. We demonstrated that J-2156 dose-dependently reduces peripheral and spinal neuronal excitability in the CFA rat model without affecting physiological pain transmission. Given the high concentration of the compound required to inhibit spinal neurons, it is unlikely that the behavioral effect seen in CFA model is mediated centrally. Overall these data demonstrated that the analgesic effect of J-2156 is mediated mainly via peripheral SST4 receptors.

CNS-infiltrating CD4+ T lymphocytes contribute to murine spinal nerve transection-induced neuropathic pain.

We previously reported leukocytic infiltration into the lumbar spinal cord in a rodent spinal nerve L5 transection (L5Tx) neuropathic pain model. Here, we further investigated the role of infiltrating T lymphocytes in the etiology of persistent pain following L5Tx. T lymphocyte-deficient nude mice showed no evident mechanical hypersensitivity after day 3 of L5Tx compared to wild-type BALB/c mice. Through FACS analysis, we determined that significant leukocytic infiltration (CD45(hi)) into the lumbar spinal cord peaked at day 7 post L5Tx. These infiltrating leukocytes contained predominantly CD4(+) but not CD8(+) T lymphocytes. B lymphocytes, natural killer cells and macrophages were not detected at day 7 post L5Tx. No differences in the activation of peripheral CD4(+) T lymphocytes were detected in either the spleen or lumbar lymph nodes between L5Tx and sham surgery groups. Further, CD4 KO mice displayed significantly decreased mechanical hypersensitivity after day 7 of L5Tx, and adoptive transfer of CD4(+) leukocytes reversed this effect. Decreased immunoreactivity of glial fibrillary acidic protein observed in CD4 KO mice post L5Tx indicated possible T lymphocyte-glial interactions. These results strongly support a contributing role of spinal cord-infiltrating CD4(+) T lymphocytes versus peripheral CD4(+) T lymphocytes in the maintenance of nerve injury-induced neuropathic pain.

Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain.

Neuroinflammatory and neuroimmune mechanisms, as exemplified by infiltrating immune cells and activation of resident endothelial/glial cells, respectively, are known to be involved in the establishment and maintenance of chronic pain. An immune system pathway that may be involved in the activation of both immune and glial cells is complement. The complement pathway is made up of a large number of distinct plasma proteins which react with one another to opsonize pathogens and induce a series of inflammatory responses to help fight infection. Cleaved products and complexes produced by complement activation are responsible for a range of effects including mediation of immune infiltration, activation of phagocytes, opsonization/lysis of pathogens and injured cells, and production of vasoactive amines such as histamine and serotonin. Gene-expression microarray-analysis performed on the rat spinal nerve ligation (SNL) model of neuropathic pain revealed that multiple complement components including the C1 inhibitor, C1q alpha, beta, and gamma, C1r, C1s, C2, C3, C4, C7, and factors B, D, H, and P, were up-regulated while DAF was down-regulated. Regulation of C3 and DAF was confirmed by real-time RT-PCR and in situ hybridization. To test the hypothesis that complement plays a role in neuropathic pain, SNL rats were treated with cobra venom factor (CVF) to deplete plasma of complement component C3. Pain behavior was significantly attenuated in SNL rats treated with CVF as was complement activity at the ipsilateral dorsal root ganglia. Our results suggest the complement pathway might be a novel target for the development of neuropathic pain therapeutics.

Biphasic form of experimental autoimmune neuritis in dark Agouti rats and its oral therapy by antigen-specific tolerization.

A new and biphasic form of experimental autoimmune neuritis (EAN) is described in dark agouti rats (DA rats) and is inducible by a single immunization with bovine peripheral nerve myelin (BPM) in complete Freund's adjuvant (DA-EAN). Animals develop a mild episode of disease; after recovery, 66-100% of the rats suffer from a more severe bout of EAN with paraparesis 25-30 days after immunization. By histology, DA-EAN is an inflammatory and demyelinating polyradiculoneuropathy virtually without axonal damage. Demyelination affects mainly spinal roots. This is also reflected by markedly increased F-wave latencies in nerve conduction studies of sciatic nerves. In sciatic nerves, inflammation and demyelination are found only focally and may be the histopathologic basis for conduction failure in some fibers. Immunologic investigations revealed stronger proliferative responses of DA than of Lewis rat lymph node cells to BPM and various peptides derived from the P2 protein. Proliferative and Th1-cytokine responses were particularly pronounced in spleen during the late phase of DA-EAN as compared to the monophasic EAN of Lewis rats. The data suggest that persistent lymphocyte proliferation with secretion of interferon (IFN)-gamma may be relevant for the relapsing course of DA-EAN whereas epitope spreading may explain the increased severity of the second bout of disease. The extended Th1 response in DA rats did not go along with a lack of downregulatory mechanisms, because the second DA-EAN attack was self-limited and splenocytes from DA rats produced considerable amounts of interleukin (IL)-10 and transforming growth factor (TGF)-beta. To substantiate further a functional immunoregulation in DA rats, we modulated DA-EAN by antigen-specific oral tolerization, which is known to involve active suppressor mechanisms. Preventive feeding of BPM in combination with cholera toxin (CT) induced a long-lasting resistance to DA-EAN. Even therapeutic administration of BPM or BPM/CT after onset of signs of disease significantly mitigated the further course of disease and prevented development of paraparesis. Because DA-EAN is easily inducible and leads consistently to relapses in most rats, it can be used for studies of immune factors that determine a relapsing course of autoimmunity. Furthermore, DA-EAN may serve as a model for relapsing inflammatory demyelinating polyneuropathies such as chronic inflammatory demyelinating polyneuropathy (CIDP) and for treatment studies. Our findings on effective prevention and therapy of DA-EAN by oral application of myelin/CT corroborate this form of immunomodulation as a treatment strategy for cell-mediated processes in chronic inflammatory neuropathies.

Presence of spinal B7.2 (CD86) but not B7.1 (CD80) co-stimulatory molecules following peripheral nerve injury: role of nondestructive immunity in neuropathic pain.

Previous work in our laboratory demonstrated spinal neuroimmune activation and leukocyte trafficking into the central nervous system (CNS) parenchyma in a rat model of neuropathic pain. Recent studies suggest that co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) play a differential role in the effect of beneficial versus deleterious CNS autoimmune responses. In the present study, we determined the lumbar spinal expression of the co-stimulatory molecules B7.1 and B7.2 in a rat model of neuropathy. We observed intense B7.2 microglial immunoreactivity in the lumbar spinal cord following the injury but no expression of B7.1. These data suggest a role of protective CNS autoimmunity following peripheral nerve injury.

Distinct functional types of macrophage in dorsal root ganglia and spinal nerves proximal to sciatic and spinal nerve transections in the rat.

Inflammation proximal to a peripheral nerve injury may be responsible for ectopic discharge and/or death of sensory neurones, factors thought to contribute to the development and/or maintenance of neuropathic pain. Here, ED1+, ED2+ and major histocompatibility complex class II (MHC II)+ macrophages in dorsal root ganglia (DRGs) and spinal nerve roots have been compared quantitatively in adult rats following transection of one sciatic or one spinal nerve, using double labelling immunohistochemistry. In control DRGs, all ED2+ cells expressed ED1 and some also MHC II. One week after either lesion, the ED2+ cells changed negligibly, except that all expressed MHC II. ED1+ and MHC II+ cell density increased markedly, with cells expressing MHC II alone (the majority), ED1/MHC II or rarely ED1 alone. In the spinal roots, ED1+ and MHC II+ cell density increased less after sciatic than after spinal nerve transection when ED1+ foamy cells were prominent. All ED2- macrophages were aggregated with T lymphocytes around blood vessels at 1 week or around isolated somata at later stages. ED1+ cell density declined more rapidly than MHC II+ cell density. Within the DRG, the debris of retrogradely labelled neurones appeared in ED2+ cells and a small proportion of MHC II+ cells that contained ED1. The data suggest that (i) resident ED2+ macrophages do not proliferate but are phagocytic and (ii) of ED1+ and MHC+ monocytes invading from the blood, only ED1+/MHC II+ cells are phagocytic. Four functional subtypes of macrophage within the DRGs were distinct from ED1+ foamy cells that phagocytosed myelin after spinal nerve transection.

Adoptive transfer-experimental allergic neuritis in newborn Lewis rats results in inflammatory infiltrates, mast cell activation, and increased Ia expression with only minor nerve fiber degeneration.

Experimental allergic neuritis (EAN) induced in the Lewis rat by the adoptive transfer of a P2-specific T cell line (AT-EAN) is considered an animal model of Guillain-Barré syndrome. It is not yet known whether AT-EAN is inducible at early stages in the development of the peripheral nervous system (PNS) or whether disease activity is modified because of immaturity of either the nervous system or the immune system. We therefore compared the susceptibility of neo-natal and adult Lewis rats to AT-EAN induced by the adoptive transfer (intraperitoneally) of 10(6) activated P2-specific T cells. P2 antigen was already present in 7 day old Lewis rats and P2-specific T cell transfer into 3-day-old rats induced clinical disease associated with an inflammatory response (sciatic nerves and spinal ganglia). In injected newborn rats we observed local activation of mast cells, infiltration of the PNS by inflammatory cells, and induction of Ia antigen expression in Schwann cells. Unlike in adults, segmental or paranodal demyelination despite occasional nerve fiber degeneration did not occur. However, the difference between newborn and adult rats could not be ascertained statistically because of the relative rarity of the lesions, their focal character, the admixture of fiber demyelination and degeneration, and most importantly, size differences of the myelinated fibers, which result in a large developmental decrease in fiber density in adults compared to newborns.

Focal peripheral nerve injury induces leukocyte trafficking into the central nervous system: potential relationship to neuropathic pain.

The present study was undertaken to determine whether leukocytes are recruited into the spinal cord following a peripheral L5 spinal nerve transection that results in mechanical allodynia (increased tactile sensitivity behavior correlates with neuropathic pain). In rats subjected to bone marrow irradiation, donor-specific major histocompatibility complex (MHC) class I (I1-69) positive peripheral immune cells trafficked to the L5 spinal cord in response to an L5 spinal nerve injury. The number of I1-69 positive cell profiles increased over time and correlated with increased mechanical allodynia. At early time points following injury, I1-69 positive immune cells co-regionalized with the expression of the macrophage marker ED2. At later time points following injury, some of the infiltrating immune cells did not co-regionalize with the macrophage marker ED2. At no time did the infiltrating cells co-regionalize with the neuronal marker (NeuN). Both macrophage-like morphology and T cell-like morphology were observed in the I1-69 positive cellular infiltrate. Conversely, animals that underwent sham surgery demonstrated little mechanical allodynia and a minimal number of infiltrating peripheral immune cells. In a separate group of rats, infiltration of CD3+ T-lymphocytes was confirmed at 14 days post-nerve transection. This study demonstrates trafficking of leukocytes into the lumbar spinal cord at time points that correlate with mechanical allodynia suggesting a role of central neuroinflammation in persistent neuropathic pain.

Altered calcium in motoneurons by IgG from human motoneuron diseases.

OBJECTIVES: The effect of IgG from patients with multifocal motor neuropathy (MMN) on the content and distribution of calcium in spinal motoneurons was compared with the effect of IgG from patients with sporadic amyotrophic lateral sclerosis (SALS) and IgG from normal individuals. MATERIAL AND METHODS: Different purified IgG samples were injected intraperitoneally in mice. Then, the animals were subjected to histochemical techniques to visualize calcium in electron microscopic sections. RESULTS: Quantitative morphometric analysis verified that IgG from MMN decreased the vesicular and axoplasmic calcium content in the axon terminals at the neuromuscular junctions and had no influence on the perikaryon. In contrast to this, IgG from patients with SALS increased the intracellular calcium both in the axon terminal and in the perikaryon. IgG from normal individuals exerted no effect. Elevated intracellular calcium may contribute to motoneuron degeneration. The lack of such effect with MMN immunoglobulins helps to explain the relative sparing of motoneurons in the disease.

Electrolytic lesion of the anterior cingulate cortex decreases inflammatory, but not neuropathic nociceptive behavior in rats.

The present study investigated the effect of lesions of the anterior cingulate cortex (ACC) on mechanical allodynia/hyperalgesia after L5 ligation or on inflammatory nociceptive responses following formalin injection in the rat. For both the neuropathic and inflammatory pain models, three groups of animals were used. The control groups consisted of a group of sham lesioned animals and a group of animals that had unilateral damage to the ACC or unilateral/bilateral damage to surrounding cortical tissue. The third group consisted of animals that had at least 75% bilateral damage of the ACC. Subjects received L5 ligation or a 0.05-ml injection of 1% formalin into the plantar surface of the hindpaw. In contrast to the control groups, bilateral ACC lesions significantly decreased inflammatory nociceptive responses during the prolonged, tonic portion of the formalin test (20-35 min). The difference between the groups was most prevalent in the amount of time spent licking the paw. However, ACC lesions did not significantly attenuate the enhanced mechanical paw withdrawal threshold in the neuropathic nociceptive model. These results suggest a differential role of the ACC in the modulation of different types of pain conditions.

Accumulation of immunoreactivity to ubiquitin carboxyl-terminal hydrolase PGP 9.5 in axons of human cases with spinal cord lesions.

The protein gene product PGP 9.5 is one of the major polypeptides in neurons. It can act as a ubiquitin carboxyl-terminal hydrolase in ubiquitin-mediated degradation of proteins. The present study was performed to find out if human cases with spinal cord trauma present immunohistochemical signs of PGP 9.5 accumulation in injured axons known to accumulate ubiquitin. For comparison, we used six autopsy cases without spinal cord pathology, one case with syringomyelia, one case with ischaemic injury of the cord, and six ALS cases. Controls presented PGP 9.5-immunostained axons of weak to moderate intensity in the longitudinal tracts. Immunoreactivity was not detected in nerve cell bodies, glial cells or axons of the grey matter. All nine trauma cases showed axonal swellings, but their numbers varied. Intensely immunostained axonal swellings were particularly abundant in cases with a survival period up to 1 month after trauma. Strongly immunoreactive axons were present also in the cases with infarct and syringomyelia. In conclusion, human cases with spinal cord trauma and other focal injuries present signs of PGP 9.5 accumulation in severed axons possibly resulting from disturbed axonal transport. PGP 9.5 thus seems to be present and may take part in ubiquitin-mediated degradation of proteins in injured axons of the spinal cord.

Amyloid neuropathy: immunocytochemical localization of intra- and extracellular immunoglobulin light chains.

Sural nerve specimens from ten patients with amyloidosis (hereditary, associated with lymphoproliferative disorders, or of unknown origin) and peripheral neuropathy were investigated by immunohistochemistry at the light and electron microscopic level. Peroxidase-antiperoxidase and immunogold techniques were applied to glutaraldehyde-fixed, osmicated and epoxy-embedded tissue. In five cases, four of which associated with lymphoproliferative disorders, amyloid deposits strongly and exclusively reacted with antibodies to kappa or lambda light chains, respectively. By electron microscopy, bundles of immunogold-labelled amyloid fibrils could be identified in coated and uncoated single membrane-bound vesicles of endoneurial macrophages. Schwann cells did not contain intracellular amyloid but their processes were entangled in amyloid fibrils and their basement membranes were sometimes fused with the fibrillar masses. It is concluded that immunoglobulin light chains in AL (amyloid of immunoglobulin light chain origin) amyloidosis precipitate, forming amyloid fibrils, in the presence of, and presumably with the assistance of, endoneurial cells. Inefficiency of phagocytosis appears to be one of the major causes for the deleterious effects of amyloid.

Binding of swine IgM immunoglobulins to peripheral nerve myelin sheaths in electron microscopic immunocytochemistry.

The IgM fraction of normal swine serum (NSS) was found to adhere to human peripheral nerve myelin sheaths from randomly selected neuropathies and control nerves in electron microscopic immunocytochemical assays. The reaction could be blocked by preincubation with undiluted fetal calf serum (FCS), whereas endoneurial background staining was already abolished by preincubation with 10% FCS. Only the IgM, but not the IgG or the IgA fraction of NSS adhered to peripheral nerve tissue. Such strong adherence of one of the components in an immunocytochemical assay may be a source of misinterpretation. Binding of swine IgM to human peripheral nerve myelin has not been described before and is of interest in the discussion about IgM binding to myelin-associated glycoprotein in IgM gammopathies.

Relationship between urotensin II- and somatostatin-immunoreactive spinal cord neurons of Catostomus commersoni and Oncorhynchus kisutch (Teleostei).

This immunocytochemical study describes the presence of separate immunoreactive (IR)-urotensin II (UII) and IR-somatostatin (SOM) systems in the spinal cord of two species of teleost fish. Both systems are arranged in a close spatial interrelationship in which IR-SOM fibres apparently innervate cerebrospinal fluid (CSF)-contacting IR-UII neurons. Specimens of Oncorhynchus kisutch also display CSF-contacting IR-SOM neurons located in the lateral ependymal walls of the central canal, in addition to CSF-contacting IR-UII neurons located ventrally. It is suggested that, in this species, CSF-contacting IR-SOM and IR-UII neurons perceive different stimuli from the CSF and are integrated in such a way that one peptidergic system may modulate the function of the other.

Direct immunofluorescence in sural nerve biopsies.

Direct immunofluorescence (DIF) has been carried out in 66 sural nerve biopsies using antibodies against human IgG, IgA, IgM, C3, C4, albumin, fibrinogen, and kappa- and lambda-chains. In 37 out of 63 (59%) different neuropathies immunoglobulins or other plasma proteins were found within the peripheral nerves. IgM along the myelin sheaths were found in monoclonal IgM-K-associated demyelinating peripheral neuropathy and chronic inflammatory demyelinating peripheral neuropathy. IgM within the perineurium were detected in hereditary, diabetic, paraneoplastic, paraproteinemic and neuropathies of unknown cause. In inflammatory, vasculitic, hereditary and toxic neuropathies fibrinogen, albumin, IgG and IgA were variably observed in endoneurium, endoneurial vessels, perineurium and epineurial vessels. In our experience DIF appears to be just an unspecific marker of sural nerve pathology. In selected cases however DIF may be helpful in the diagnosis or in better understanding the pathogenetic mechanisms of the disease.

Class II antigen expression and inflammatory cells in the Guillain-Barré syndrome.

Monoclonal antibodies to T-lymphocyte subsets and Class II molecules (Ia) have been used to characterize the inflammatory infiltrate in nerve tissue biopsied from 2 patients in the acute phase of Guillain-Barré syndrome; the findings were compared with those in control nerve specimens. Normal control nerve was treated in the same way. In normal nerves, Class II molecules are expressed on endothelial cells and on occasional mononuclear and perineurial cells. In Guillain-Barré nerves the inflammatory infiltrate consisted mainly of Class II-positive cells of the monocyte-macrophage lineage and of lesser numbers of T4 and T8 lymphoid cells. T4 cells predominated in perivascular collections. In the more severely affected patient, there was a marked increase of Class II molecules expressed on endothelial cells, perineurial cells, and most Schwann cells. Schwann cells associated with unmyelinated fibers and the Schwann cell processes of denervated Büngner bands all expressed Class II molecules. These histological changes were less marked in the more mildly affected patient. It is suggested that the expression of Class II molecules on the myelin forming cell, the Schwann cell, has important implications for the pathogenesis of the demyelination that occurs in Guillain-Barré syndrome.