Cecilia A. Dominguez

Multipotent mesenchymal stromal cells attenuate chronic inflammation and injury-induced sensitivity to mechanical stimuli in experimental spinal cord injury.

PURPOSE Previous reports established that after a contusion injury to the rat spinal cord, locomotor function was enhanced by the transplantation of cells from bone marrow referred to as either mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). It has also been established that neural stem cells (NSCs) enhance locomotor function after transplantation into the injured rat spinal cord. However, the beneficial effects of NSCs are limited by graft-induced allodynia-like responses. Little is known about the effects of MSCs on sensory function in spinal cord injury. Therefore, the objective of this research was to determine whether transplantation of MSCs into the injured rat spinal cord induces allodynia-like responses. METHODS Contusion injuries of two different severities were induced in rats to examine the effects of transplantation with MSCs on sensorimotor deficits. The effects of MSCs on chronic inflammation were investigated, since inflammation is reported to have a role in the sensorimotor deficits associated with spinal cord injury. In addition, observations in other models suggest that MSCs possess immunosuppressive effects. RESULTS We found that in contrast to previous observations with the transplantation of neural stem cells, transplantation of MSCs did not induce allodynia. MSCs attenuated injury-induced sensitivity to mechanical stimuli but had no effect on injury-induced sensitivity to cold stimuli. MSCs also significantly attenuated the chronic inflammatory response as assayed by GFAP immunoreactivity for reactive astrocytes and ED1 immunoreactivity for activated macrophages/microglia. In addition, transplantation of MSCs increased white matter volumes and decreased cyst size in sections of the cord containing the lesion. CONCLUSION The results suggest that the sensorimotor enhancements produced by MSCs can at least in part be explained by anti-inflammatory/immunosuppressive effects of the cells, similar to such effects of these cells observed in other experimental models.

Spontaneous pain following spinal nerve injury in mice

Autotomy behavior is frequently observed in rats and mice in which the nerves of the hindlimb are severed, denervating the paw. This is the neuroma model of neuropathic pain. A large body of evidence suggests that this behavior reflects the presence of spontaneous dysesthesia and pain. In contrast, autotomy typically does not develop in partial nerve injury pain models, leading to the belief that these animals develop hypersensibility to applied stimuli (allodynia and hyperalgesia), but not spontaneous pain. We have modified the widely used Chung (spinal nerve ligation [SNL]) model of neuropathic pain in a way that retains the fundamental neural lesion, but eliminates nociceptive sensory cover of the paw. These animals performed autotomy. Moreover, the heritable across strains predisposition to spontaneous pain behavior in this new proximal denervation model (SNN) was highly correlated with pain phenotype in the neuroma model suggesting that the pain mechanism in the two models is the same. Relative reproducibility of strain predispositions across laboratories was verified. These data indicate that the neural substrate for spontaneous pain is present in the Chung-SNL model, and perhaps in the other partial nerve injury models as well, but that spontaneous pain is not expressed as autotomy in these models because there is protective nociceptive sensory cover.

Genetic analysis of neuropathic pain-like behavior following peripheral nerve injury suggests a role of the major histocompatibility complex in development of allodynia.

&NA; Neuropathic pain is a common consequence of damage to the nervous system. We here report a genetic analysis of development of neuropathic pain‐like behaviors after unilateral photochemically‐induced ischemic sciatic nerve injury in a panel of inbred rat strains known to display different susceptibility to autoimmune neuroinflammation. Pain behavior was initially characterized in Dark‐Agouti (DA; RT1av1), Piebald Virol Glaxo (PVG; RT1c), and in the major histocompatibility complex (MHC)‐congenic strain PVG‐RT1av1. All strains developed mechanical hypersensitivity (allodynia) following nerve injury. However, the extent and duration of allodynia varied significantly among the strains, with PVG displaying more severe allodynia compared to DA rats. Interestingly, the response of PVG‐RT1avRT1 was similar to that of DA, suggesting regulation by the MHC locus. This notion was subsequently confirmed in an F2 cohort derived from crossing of the PVG and PVG‐RT1av1strains, where allodynia was reduced in homozygous or heterozygous carriers of the RT1av1 allele in comparison to rats homozygous for the RT1c allele. These results indicate that certain allelic variants of the MHC could influence susceptibility to develop and maintain neuropathic pain‐like behavior following peripheral nerve injury in rats.

The DQB1*03:02 HLA haplotype is associated with increased risk of chronic pain after inguinal hernia surgery and lumbar disc herniation

Summary There is a human leukocyte antigen‐dependent risk of developing neuropathic pain after inguinal surgery and it is mediated by the DRB1*04‐DQB1*03:02 haplotype. ABSTRACT Neuropathic pain conditions are common after nerve injuries and are suggested to be regulated in part by genetic factors. We have previously demonstrated a strong genetic influence of the rat major histocompatibility complex on development of neuropathic pain behavior after peripheral nerve injury. In order to study if the corresponding human leukocyte antigen complex (HLA) also influences susceptibility to pain, we performed an association study in patients that had undergone surgery for inguinal hernia (n = 189). One group had developed a chronic pain state following the surgical procedure, while the control group had undergone the same type of operation, without any persistent pain. HLA DRB1genotyping revealed a significantly increased proportion of patients in the pain group carrying DRB1*04 compared to patients in the pain‐free group. Additional typing of the DQB1 gene further strengthened the association; carriers of the DQB1*03:02 allele together with DRB1*04 displayed an increased risk of postsurgery pain with an odds risk of 3.16 (1.61‐6.22) compared to noncarriers. This finding was subsequently replicated in the clinical material of patients with lumbar disc herniation (n = 258), where carriers of the DQB1*03:02 allele displayed a slower recovery and increased pain. In conclusion, we here for the first time demonstrate that there is an HLA‐dependent risk of developing pain after surgery or lumbar disc herniation; mediated by the DRB1*04 – DQB1*03:02 haplotype. Further experimental and clinical studies are needed to fine‐map the HLA effect and to address underlying mechanisms.

Recovery from spinal cord injury differs between rat strains in a major histocompatibility complex-independent manner

Inflammation is a common characteristic of spinal cord injury. The nature of this response, whether it is beneficial or detrimental, has been the subject of debate. It has been reported that susceptibility to autoimmunity is correlated with increased functional impairment following spinal cord injury. As the ability to mount an autoimmune response has most consistently been associated with certain haplotypes of the major histocompatibility complex (MHC), we analysed the possible effects of the MHC haplotype on functional impairment and recovery following spinal cord injury. A contusion injury was induced in experimental autoimmune encephalomyelitis‐susceptible and ‐resistant rats [Dark Agouti, Lewis and Piebald Viral Glaxo (PVG), respectively]. We found that locomotion recovered significantly better in Dark Agouti rats compared with PVG and Lewis rats but an F2 intercross (PVG × PVG‐RT1av1) excluded the possibility that this difference was MHC haplotype‐dependent. Thus, we conclude that recovery following spinal cord injury is subject to considerable genetic heterogeneity that is not coupled to the MHC haplotype region. Continued research of genetic variants regulating recovery following spinal cord injury is warranted.

Sex differences in the development of localized and spread mechanical hypersensitivity in rats after injury to the infraorbital or sciatic nerves to create a model for neuropathic pain

BACKGROUND Neuropathic pain after injury to the nervous system is a difficult clinical problem. Sex differences in the development of neuropathic pain have not been well established experimentally or clinically. OBJECTIVE Rats were used to examine sex differences in localized and spread mechanical hypersensitivity after partial injury to their infraorbital or sciatic nerves in a model of neuropathic pain. METHODS In adult female and male rats, partial nerve injury to the infraorbital and sciatic nerves was produced using a photochemical method. Mechanical hypersensitivity (allodynia) was examined and compared in the innervation territories of the nerves on the face or hind paw. The spread of hypersensitivity beyond the innervation territories of the injured nerves was also studied. The female and male rats were randomized to active and sham groups. The rats in the sham group had their sciatic or infraorbital nerve exposed, but not injured. RESULTS A total of 67 rats (36 females, 31 males) were used. There was a marked sex difference in the response to infraorbital nerve injury: female rats developed more profound and long-lasting facial hypersensitivity than did male rats (P<0.001). Hypersensitivity of the hind paw after sciatic nerve injury did not, however, significantly differ between female and male rats. Spread mechanical hypersensitivity was noted in body areas outside the innervation territory of the injured nerve. This hypersensitivity was more profound after infraorbital than sciatic nerve injury and also displayed a significant sex difference (female>male, P < 0.001). Sham-group rats did not exhibit localized or spread mechanical hypersensitivity. CONCLUSION Sex differences in the development of neuropathic painlike behaviors in rats were dependent on site of injury and site of testing, with female rats being more susceptible to the development of spread mechanical hypersensitivity, particularly after facial nerve injury, compared with male rats.

Cranial irradiation induces transient microglia accumulation, followed by long-lasting inflammation and loss of microglia

The relative contribution of resident microglia and peripheral monocyte-derived macrophages in neuroinflammation after cranial irradiation is not known. A single dose of 8 Gy was administered to postnatal day 10 (juvenile) or 90 (adult) CX3CR1GFP/+ CCR2RFP/+ mouse brains. Microglia accumulated in the subgranular zone of the hippocampal granule cell layer, where progenitor cell death was prominent. The peak was earlier (6 h vs. 24 h) but less pronounced in adult brains. The increase in juvenile, but not adult, brains was partly attributed to proliferation. Microglia numbers then decreased over time to 39% (juvenile) and 58% (adult) of controls 30 days after irradiation, largely as a result of cell death. CD68 was expressed in 90% of amoeboid microglia in juvenile hippocampi but only in 9% of adult ones. Isolated hippocampal microglia revealed reduced CD206 and increased IL1-beta expression after irradiation, more pronounced in juvenile brains. CCL2 and IL-1 beta increased after irradiation, more in juvenile hippocampi, and remained elevated at all time points. In summary, microglia activation after irradiation was more pronounced, protracted and pro-inflammatory by nature in juvenile than in adult hippocampi. Common to both ages was long-lasting inflammation and the absence of monocyte-derived macrophages.

Unbiased Expression Mapping Identifies a Link between the Complement and Cholinergic Systems in the Rat Central Nervous System

The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.

The DQB1(∗)03:02 HLA haplotype is associated with increased risk of chronic pain after inguinal hernia surgery and lumbar disc herniation

Abstract Neuropathic pain conditions are common after nerve injuries and are suggested to be regulated in part by genetic factors. We have previously demonstrated a strong genetic influence of the rat major histocompatibility complex on development of neuropathic pain behavior after peripheral nerve injury. In order to study if the corresponding human leukocyte antigen complex (HLA) also influences susceptibility to pain, we performed an association study in patients that had undergone surgery for inguinal hernia (n = 189). One group had developed a chronic pain state following the surgical procedure, while the control group had undergone the same type of operation, without any persistent pain. HLA DRB1genotyping revealed a significantly increased proportion of patients in the pain group carrying DRB1(*)04 compared to patients in the pain-free group. Additional typing of the DQB1 gene further strengthened the association; carriers of the DQB1(*)03:02 allele together with DRB1(*)04 displayed an increased risk of postsurgery pain with an odds risk of 3.16 (1.61-6.22) compared to noncarriers. This finding was subsequently replicated in the clinical material of patients with lumbar disc herniation (n = 258), where carriers of the DQB1(*)03:02 allele displayed a slower recovery and increased pain. In conclusion, we here for the first time demonstrate that there is an HLA-dependent risk of developing pain after surgery or lumbar disc herniation; mediated by the DRB1(*)04-DQB1(*)03:02 haplotype. Further experimental and clinical studies are needed to fine-map the HLA effect and to address underlying mechanisms.

Genetic and sex influence on neuropathic pain-like behaviour after spinal cord injury in the rat

Chronic pain of neuropathic nature after spinal cord injury (SCI) is common and its underlying mechanisms are poorly understood. Genes, as well as sex, have been implicated, but not thoroughly investigated in experimental genetic models for complex traits. We have previously found that inbred Dark‐Agouti (DA) rats develop more severe SCI pain‐like behaviour than a major histocompatibility complex‐congenic Piebald Virol Glaxo (PVG)‐RT1av1 strain in a model of photochemically induced SCI.