Brian J. MacNeil

Elevated Expression of Fractalkine (CX3CL1) and Fractalkine Receptor (CX3CR1) in the Dorsal Root Ganglia and Spinal Cord in Experimental Autoimmune Encephalomyelitis: Implications in Multiple Sclerosis-Induced Neuropathic Pain

Multiple sclerosis (MS) is a central nervous system (CNS) disease resulting from a targeted autoimmune-mediated attack on myelin proteins in the CNS. The release of Th1 inflammatory mediators in the CNS activates macrophages, antibodies, and microglia resulting in myelin damage and the induction of neuropathic pain (NPP). Molecular signaling through fractalkine (CX3CL1), a nociceptive chemokine, via its receptor (CX3CR1) is thought to be associated with MS-induced NPP. An experimental autoimmune encephalomyelitis (EAE) model of MS was utilized to assess time dependent gene and protein expression changes of CX3CL1 and CX3CR1. Results revealed significant increases in mRNA and the protein expression of CX3CL1 and CX3CR1 in the dorsal root ganglia (DRG) and spinal cord (SC) 12 days after EAE induction compared to controls. This increased expression correlated with behavioural thermal sensory abnormalities consistent with NPP. Furthermore, this increased expression correlated with the peak neurological disability caused by EAE induction. This is the first study to identify CX3CL1 signaling through CX3CR1 via the DRG /SC anatomical connection that represents a critical pathway involved in NPP induction in an EAE model of MS.

Contribution of the adrenal glands and splenic nerve to LPS-induced splenic cytokine production in the rat.

Both the hypothalamic pituitary adrenal axis (HPAA) and the sympathetic nervous system (SNS) can inhibit immune function and are regarded as the primary efferent pathways for neural-immune interactions. To determine if this relationship is maintained in vivo in response to an inflammatory stimulus, rats were injected intravenously (iv) with various doses of lipopolysaccharide (LPS) and splenic cytokine mRNA and protein levels were measured at several dose and time intervals post-injection. The spleen was chosen as the target organ because both the neural and hormonal inputs to the spleen can be selectively removed by splenic nerve cut (SNC) and adrenalectomy (ADX), respectively. Data from our dose response studies established that maximum levels of splenic cytokines were induced in response to relatively low doses of LPS. Minimal changes in LPS-induced splenic cytokine levels were observed in response to ADX, SNC, or a combination of the two procedures across several doses of LPS. These results suggest that there are aspects of immune regulation that are functionally removed from these central modulatory systems and that the counter-regulatory responses induced by LPS have minimal impact on the concurrent induction of cytokines by this inflammatory stimulus. The conceptual model of neural-immune regulation as an inhibitory feedback system, at least with regards to the early activational effects induced by an inflammatory stimulus, was not supported by these studies.

The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain‐derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post‐disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG‐derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.

Immunoregulation by the sympathetic nervous system

Abstract The neuroendocrine and autonomic nervous systems constitute efferent pathways through which the nervous system modulates peripheral immune responses. Regulation of glucocorticoid levels by the HPA axis is a primary component of the neural-immune regulatory system, whereas the role of the autonomic nervous system in the regulation of immune function is not fully elucidated. We have identified two paradigms, central inflammatory stimuli and stress, for which an immunosuppressive role of the sympathetic nervous system has been demonstrated. Since the spleen is exclusively innervated by sympathetic nerve fibers and is accessible for experimental manipulation, we have utilized this secondary immune organ as a model system for analyzing brain-immune interactions. Central injections of inflammatory stimuli (IL-1 or PGE2), as well as stress, produce an acute suppression of splenic macrophage function. Although the HPA axis and the sympathetic nervous system are jointly activated by these treatments, we have shown that the acute suppression of splenic macrophage function by central inflammatory stimuli and stress are still observed in adrenalectomized animals. Abrogation of this adrenal-independent immunosuppression in splenic immune function by surgically cutting the sympathetic nerve fibers innervating the spleen illustrates that the sympathetic nervous system constitutes an important pathway for the neural regulation of peripheral immune function. Although both stress and immune stimuli activate the same efferent system, they access this regulatory system via different neural pathways. The paraventricular nucleus (PVN) is proposed as an essential component of this regulatory network and a nodal region for the ingegration and regulation of both neuroendocrine and autonomic responses. Brain stem knife cuts or posterolateral deafferentation of the PVN indicate that activation of the PVN by immune stimuli are primarily, if not exclusively, mediated by ascending brain stem afferents to the PVN. stimuli are primarily, if not exclusively, mediated by ascending brain stem afferents to the PVN. These same brain stem knife cuts have a minimal effect on the activation of the PVN by stress and loss of posterior and lateral connections of the PVN only partially attenuates the activation of the PVN by stress. These results indicate that, in contrast to immune related, stimuli, rostral inputs to the PVN mediate a major portion of the activational effects of stress on the PVN. Thus, the HPA axis and the sympathetic nervous system are the two primary output pathways utilized by the neural-immune regulatory system to regulate peripheral immune responses.

The effects of sildenafil on the cardiovascular response in men with spinal cord injury at or above the sixth thoracic level.

Abstract Background: Sildenafil is efficacious for erectile dysfunction in men with spinal cord injury (SCI), but can induce hypotension in neurologically intact people. Those with SCI at or above the sixth thoracic level (T6) often have pre-existing hypotension, yet the cardiovascular response to sildenafil has not been studied in this group. Objective: To evaluate the effect of sildenafil on the cardiovascular response in men with complete SCI at or above T6. Methods: This was a randomized, double-blind, placebo-controlled, cross-over study. Twenty-three SCI participants were each randomly given placebo; sildenafil, 50 mg; and sildenafil, 1 00 mg; separated by at least 1 week. The following were measured before administration, and hourly for 4 hours afterward: (a) blood pressure (BP) and heart rate (HR) , both supine and sitting; and (b) perceived dizziness on a visual analog scale upon sitting. Results: Analysis was done using a 4-way repeated-measures analysis of variance. No significant changes occurred with placebo. Sildenafil caused the following changes. Systolic BP changed little in thoracic spinal cord-injured (TSCI) participants, but decreased significantly (P < 0.005) in cervical spinal cord-injured (CSCI) participants. Diastolic BP decreased in all participants (P < 0.005). HR increased in the TSCI participants for 1 hour (P < 0.05), but was not altered in the CSCI participants. Dizziness increased in the TSCI participants after administration of 100 mg (P < 0.05) and in the CSCI participants after administration of 50 mg (P < 0.05). There were no adverse events or outcomes. Conclusion: Sildenafil induces significant hypotension in people with cervical-level injuries-more so than in thoracic-level injuries-and can cause dizziness in both populations. It should be prescribed with caution and informed consent from the patient.

Neurochemical excitation of thoracic propriospinal neurons improves hindlimb stepping in adult rats with spinal cord lesions

Using an in vitro neonatal rat brainstem-spinal cord preparation, we previously showed that cervicothoracic propriospinal neurons contribute to descending transmission of the bulbospinal locomotor command signal, and neurochemical excitation of these neurons facilitates signal propagation. The present study examined the relevance of these observations to adult rats in vivo. The first aim was to determine the extent to which rats are able to spontaneously recover hindlimb locomotor function in the presence of staggered contralateral hemisections (left T2-4 and right T9-11) designed to abolish all long direct bulbospinal projections. The second aim was to determine whether neurochemical excitation of thoracic propriospinal neurons in such animals facilitates hindlimb stepping. In the absence of intrathecal drug injection, all animals (n=24) displayed some degree of hindlimb recovery ranging from weak ankle movements to brief periods of unsupported hindlimb stepping on the treadmill. The effect of boluses of neurochemicals delivered via an intrathecal catheter (tip placed midway between the rostral and caudal thoracic hemisections) was examined at post-lesion weeks 3, 6 and 9. Quipazine was particularly effective facilitating hindlimb stepping. Subsequent complete transection above the rostral (n=3) or caudal (n=2) hemisections at week 9 had no consistent effect on drug-free locomotor performance, but the facilitatory effect of drug injection decreased in 4/5 animals. Two animals underwent complete transection at T3 as the first and only surgery and implantation of two intrathecal catheters targeted to the mid-thoracic and lumbar regions, respectively. A similar facilitatory effect on stepping was observed in response to drugs administered via either catheter. The results indicate that partial spontaneous recovery of stepping occurs in adult rats after abolishing all long direct bulbospinal connections, in contrast to previous studies suggesting that hindlimb stepping after dual hemisections either does not occur or is observed only if the second hemisection surgery is delayed relative to the first. The results support the hypothesis that artificial modulation of propriospinal neuron excitability may facilitate recovery of motor function after spinal cord injury. However, whether this facilitation is due to enhanced transmission of a descending locomotor signal or is the result of excitation of thoracolumbar circuits independent of supraspinal influence, requires further study.

Diverse sensory inputs permit priming in the acidic saline model of hyperalgesia

The rodent acidic saline model of hyperalgesia uses repeat injections of acidic saline in the right lateral gastrocnemius muscle, spaced five days apart, to induce a persistent decrease in hindpaw withdrawal thresholds. The objective of this study was to determine if alternate injection sites would permit development of hyperalgesia.

Maternal distress in early life predicts the waist-to-hip ratio in schoolchildren

We report on life course stress determinants of overweight in children, using data from the longitudinal follow-up of the nested case-control arm of the SAGE (study of asthma genes and the environment) birth cohort in Manitoba, Canada. Waist and hip measurements were obtained during a clinic visit at age 9-11 years. Multiple linear regression was conducted to determine the relationship between the waist-to-hip ratio and maternal smoking during pregnancy, postpartum maternal distress and stress reactivity in children (cortisol, cortisol-DHEA [dihydroepiandrostrenone] ratio quartiles) following a clinic stressor at age 8-10 years. We found waist-to-hip risk at age 9-11 years to be elevated among boys and girls whose mothers had experienced distress in the postnatal period. This association varied by gender and asthma status. In healthy girls, postpartum distress increased waist-to-hip ratio by a factor of 0.034 (P < 0.01), independent of the childs stage of puberty and adrenarche, cortisol-DHEA ratio and duration of exclusive breastfeeding. Among girls with asthma, maternal smoking during pregnancy was associated with an increased waist-to-hip ratio, if the mother also experienced distress in the postpartum period (0.072, P = 0.038). Among asthmatic boys, an association between maternal distress and waist-to-hip ratio was evident at the highest cortisol-DHEA ratios. Stress-induced changes to leptin and infant over-eating pathways were proposed to explain the postnatal maternal distress effects. Drawing on the theories of evolutionary biology, our findings underscore the significance of postnatal stress in disrupting hypothalamic-pituitary-adrenal axis function in infants and increasing risk for child overweight.

Skin inflammation and immunity after spinal cord injury

Abtract Spinal cord injury (SCI) is associated with an elevated risk of infection as well as increased mortality from septicemia. Pressure ulcers of the skin, a frequent complication after SCI, are a major source of these infections. Reports of suppressed immune function in otherwise healthy SCI humans suggests that altered immunity may be part of the sequela of SCI. Therefore, we examined whether impaired inflammation and immune function could be demonstrated in an animal model of complete SCI. In an initial study, rats given a complete SCI at the T1–T2 level were challenged with lipopolysaccharide (LPS) 2 weeks after the SCI. In the spleen, production of tumor necrosis factor-alpha (TNF-α) mRNA and protein were elevated in SCI rats above that of control animals whereas interleukin-1 beta (IL-1β) synthesis was not effected. These changes were specific to the spleen, an immune organ, since production of these cytokines in the liver was not effected by SCI. To extend these studies to the site of antigen entry at pressure ulcers, we tested how well the skin of SCI rats could generate an inflammatory response. Injection of turpentine into the skin produced robust inflammation in control animals which was fully established tow hours after the injection. In contrast, SCI rats showed little or no sign of inflammation until 12–24 hours after the injection. The cutaneous inflammatory response to turpentine was further characterized by measuring cytokine production two hours after the injection. A significant reduction in the synthesis of TNF-α, IL-1β, IL-6, and MCP were seen below the level of the SCI compared to skin from the same site in control rats. These results demonstrate that the changes in skin immune function are limited to skin which is affected by the SCI. We propose that dysregulated neural input to skin following SCI is a primary mediator of the altered inflammatory response in skin and may increase susceptibility to infection.

Spinal Cord Brain Derived Neurotrophic Factor (BDNF) Responsive Cells in an Experimental Autoimmune Encephalomyelitis (EAE) Model of Multiple Sclerosis (MS): Implications in Myelin Repair

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease that destroys central nervous system (CNS) myelin. Although, the exact pathophysiology of MS is unknown, it is associated with CNS infiltration of T-cells and monocytes, which subsequently activate phagocytic cells that directly damage myelin. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase receptor (TrkB), have recognized roles in myelin structure formation, maintenance, and repair. We used an experimental autoimmune encephalomyelitis (EAE) model of MS to determine changes in TrkB expression that may contribute to neurological recovery and myelin repair following an early inflammatory immune-mediated attack on CNS myelin. Spinal cord (SC) TrkB gene and protein expression were analyzed at various time intervals post-EAE induction. Analysis of gene and protein expression was conducted in animals with EAE relative to active controls (AC) and naive controls (NC). We showed significant increases in TrkB protein in the SC of EAE rats 12 days post-induction relative to controls. This elevated TrkB expression correlated with the onset of neurological recovery days 12 to 15 post-EAE induction. Furthermore, immunohistochemistry (IHC) analysis revealed up-regulated expression of TrkB in several SC cell types including a specific subset of BDNF responsive neuronal cells. Finally, transmission electron microscopy (TEM) showed the ultrastructural integrity of myelin is already compromised during the early, inflammatory stage of EAE prior to widespread demyelination. Therefore, the molecular signaling of SC BDNF via TrkB represents a key therapeutic target whose manipulation could facilitate myelin repair and neurological recovery following an MS-induced myelin attack.