Gustavo Tenorio

Resident glial cell activation in response to perispinal inflammation leads to acute changes in nociceptive sensitivity: Implications for the generation of neuropathic pain

Summary Inflammation of the dorsal spinal cord leads to significant but short‐lived changes in nociceptive sensory thresholds due in part to the maintenance of the blood–spinal cord barrier. Abstract Injury or disease affecting the spinal cord is often accompanied by abnormal, chronic pain. Recent estimates suggest that approximately 60% of patients with multiple sclerosis are affected by significant changes in pain sensitivity or experience ongoing neuropathic pain of unknown etiology. Chronic pain is also a significant concern after direct spinal cord trauma. Inflammatory events and the changes in astrocyte and microglia reactivity at the spinal level in response to injury or disease are now recognized as important processes that can initiate pain hypersensitivity. Changes in the structural integrity or permeability of the blood–brain barrier/blood–spinal cord barrier (BBB/BSCB) can facilitate the inflammatory events that result in these abnormal pain states. It remains unclear, however, whether chronic pain in these disorders is dependent on the influx of peripheral leukocytes or whether changes in the reactivity of resident glial cells within the central nervous system alone are sufficient. To address this question, we generated a model of perispinal inflammation that resulted in significant changes in the reactivity of resident astrocytes and microglia within the spinal cord but maintained the integrity of the BSCB. A number of similar changes at the behavioural and cellular level occur in this model that mimic the responses seen in animal models of multiple sclerosis or spinal cord injury (SCI). However, these changes are short lived and resolve over the course of a 2‐week observation period. Our findings suggest that the chronicity of pain after injury or disease in the nervous system is dependent on the integrity of the BBB/BSCB.

A diminished response to formalin stimulation reveals a role for the glutamate transporters in the altered pain sensitivity of mice with experimental autoimmune encephalomyelitis (EAE).

&NA; Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) in which neuropathic pain is now recognized as a major symptom. To date, few studies have examined the underlying mechanisms of neuropathic pain in MS. Recently we showed that in a chronic‐relapsing animal model of MS, experimental autoimmune encephalomyelitis (EAE), characteristic neuropathic behaviours develop. However, responses to persistent noxious stimuli in EAE remain unexplored. We, therefore set out to characterize the changes in pain sensitivity in our EAE model to subcutaneous injection of formalin. We show here that female C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG35–55) display a significant decrease in elicited pain behaviours in response to formalin injection. These effects were found to involve dysregulation of the glutamatergic system in EAE. We show here that these effects are mediated by decreased glutamate transporter expression associated with EAE. Our findings demonstrate that dysregulation of glutamate transporter function in EAE mice is an important mechanism underlying the abnormal pain sensitivity in response to persistent noxious stimulation of mice with EAE and also sheds light on a potential mechanism underlying neuropathic pain behaviours in this model.

Changes in nociceptive sensitivity and object recognition in experimental autoimmune encephalomyelitis (EAE).

Multiple sclerosis is associated with a high incidence of depression, cognitive impairments and neuropathic pain. Previously, we demonstrated that tactile allodynia is present at disease onset in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). We have now monitored changes in object recognition in mice with EAE to determine if altered nociceptive sensitivity is also associated with behavioral signs indicative of cognitive impairment in this model. At the onset of clinical signs, mice with EAE showed impairments in the novel object recognition (NOR) assay, indicative of deficits in cognitive functioning early in the disease course. At the spinal level, we found increased gene expression for the cytokines IL-1β, IL-6 and the glutamate transporter EAAT-2 that coincide with increased nociceptive sensitivity and deficits in object recognition. Increased levels of EAAT-2 mRNA appear to be a response to perturbed protein levels of the transporter as we found a loss of EAAT-2 protein levels in the spinal cord of EAE mice. To determine if changes in the levels of EAAT-2 were responsible for the observed changes in nociceptive sensitivity and cognitive deficits, we treated EAE mice with the β-lactam antibiotic ceftriaxone, an agent known to increase glutamate transporter levels in vivo. Ceftriaxone prevented tactile hypersensitivity and normalized performance in the NOR assay in EAE mice. These findings highlight the important interrelationship between pain and cognitive function in the disease and suggest that targeting spinally mediated pain hypersensitivity is a novel therapeutic avenue to treat impairments in other higher order cortical processes.

Voluntary wheel running delays disease onset and reduces pain hypersensitivity in early experimental autoimmune encephalomyelitis (EAE).

Multiple sclerosis (MS) is classically defined by motor deficits, but it is also associated with the secondary symptoms of pain, depression, and anxiety. Up to this point modifying these secondary symptoms has been difficult. There is evidence that both MS and the animal model experimental autoimmune encephalomyelitis (EAE), commonly used to study the pathophysiology of the disease, can be modulated by exercise. To examine whether limited voluntary wheel running could modulate EAE disease progression and the co-morbid symptoms of pain, mice with EAE were allowed access to running wheels for 1h every day. Allowing only 1h every day of voluntary running led to a significant delay in the onset of clinical signs of the disease. The development of mechanical allodynia was assessed using Von Frey hairs and indicated that wheel running had a modest positive effect on the pain hypersensitivity associated with EAE. These behavioral changes were associated with reduced numbers of cFOS and phosphorylated NR1 positive cells in the dorsal horn of the spinal cord compared to no-run EAE controls. In addition, within the dorsal horn, voluntary wheel running reduced the number of infiltrating CD3(+) T-cells and reduced the overall levels of Iba1 immunoreactivity. Using high performance liquid chromatography (HPLC), we observed that wheel-running lead to significant changes in the spinal cord levels of the antioxidant glutathione. Oxidative stress has separately been shown to contribute to EAE disease progression and neuropathic pain. Together these results indicate that in mice with EAE, voluntary motor activity can delay the onset of clinical signs and reduce pain symptoms associated with the disease.

Tissue concentration changes of amino acids and biogenic amines in the central nervous system of mice with experimental autoimmune encephalomyelitis (EAE).

We have characterized the changes in tissue concentrations of amino acids and biogenic amines in the central nervous system (CNS) of mice with MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model commonly used to study multiple sclerosis (MS). High performance liquid chromatography was used to analyse tissue samples from five regions of the CNS at the onset, peak and chronic phase of MOG(35-55) EAE. Our analysis includes the evaluation of several newly examined amino acids including d-serine, and the inter-relations between the intraspinal concentration changes of different amino acids and biogenic amines during EAE. Our results confirm many of the findings from similar studies using different variants of the EAE model as well as those examining changes in amino acid and biogenic amine levels in the cerebrospinal fluid (CSF) of MS patients. However, several notable differences were observed between mice with MOG(35-55)-induced EAE with findings from human studies and other EAE models. In addition, our analysis has identified strong correlations between different amino acids and biogenic amines that appear to change in two distinct groups during EAE. Group I analyte concentrations are increased at EAE onset and peak but then decrease in the chronic phase with a large degree of variability. Group II is composed of amino acids and biogenic amines that change in a progressive manner during EAE. The altered levels of these amino acids and biogenic amines in the disease may represent a critical pathway leading to neurodegenerative processes that are now recognized to occur in EAE and MS.

Altered excitatory-inhibitory balance within somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a mouse model of multiple sclerosis

BackgroundChronic neuropathic pain is a common symptom of multiple sclerosis (MS). MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) has been used as an animal model to investigate the mechanisms of pain in MS. Previous studies have implicated sensitization of spinal nociceptive networks in the pathogenesis of pain in EAE. However, the involvement of supraspinal sites of nociceptive integration, such as the primary somatosensory cortex (S1), has not been defined. We therefore examined functional, structural, and immunological alterations in S1 during the early stages of EAE, when pain behaviors first appear.We also assessed the effects of the antidepressant phenelzine (PLZ) on S1 alterations and nociceptive (mechanical) sensitivity in early EAE. PLZ has been shown to restore central nervous system (CNS) tissue concentrations of GABA and the monoamines (5-HT, NA) in EAE. We hypothesized that PLZ treatment would also normalize nociceptive sensitivity in EAE by restoring the balance of excitation and inhibition (E-I) in the CNS.MethodsWe used in vivo flavoprotein autofluorescence imaging (FAI) to assess neural ensemble responses in S1 to vibrotactile stimulation of the limbs in early EAE. We also used immunohistochemistry (IHC), and Golgi-Cox staining, to examine synaptic changes and neuroinflammation in S1. Mechanical sensitivity was assessed at the clinical onset of EAE with Von Frey hairs.ResultsMice with early EAE exhibited significantly intensified and expanded FAI responses in S1 compared to controls. IHC revealed increased vesicular glutamate transporter (VGLUT1) expression and disrupted parvalbumin+ (PV+) interneuron connectivity in S1 of EAE mice. Furthermore, peri-neuronal nets (PNNs) were significantly reduced in S1. Morphological analysis of excitatory neurons in S1 revealed increased dendritic spine densities. Iba-1+ cortical microglia were significantly elevated early in the disease. Chronic PLZ treatment was found to normalize mechanical thresholds in EAE. PLZ also normalized S1 FAI responses, neuronal morphologies, and cortical microglia numbers and attenuated VGLUT1 reactivity—but did not significantly attenuate the loss of PNNs.ConclusionsThese findings implicate a pro-excitatory shift in the E-I balance of the somatosensory CNS, arising early in the pathogenesis EAE and leading to large-scale functional and structural plasticity in S1. They also suggest a novel antinociceptive effect of PLZ treatment.

Protein kinase C gamma (PKCγ) as a novel marker to assess the functional status of the corticospinal tract in experimental autoimmune encephalomyelitis (EAE)

In the spinal cord, PKCγ is an important kinase found in a specific subset of excitatory interneurons in the superficial dorsal horn and in axons of the corticospinal tract (CST). The major interest in spinal PKCγ has been its influences on regulating pain sensitivity but its presence in the CST also indicates that it has a significant role in locomotor function. A hallmark feature of the animal model commonly used to study Multiple Sclerosis, experimental autoimmune encephalolomyelitis (EAE) are motor impairments associated with the disease. More recently, it has also become recognized that EAE is associated with significant changes in pain sensitivity. Given its role in generating pain hypersensitivity and its presence in a major tract controlling motor activity, we set out to characterize whether EAE was associated with changes PKCγ levels in the spinal cord. We show here that EAE triggers a significant reduction in the levels of PKCγ, primarily in the CST. We did not observe any significant changes in PKCγ levels in the superficial dorsal horn but in general the levels tended to be below control levels in this region. In a final experiment we assessed the levels of PKCγ in the spinal cord of EAE mice that had recovered gross locomotor function and compared this to the levels found in EAE mice with chronic deficits. Our findings demonstrate that PKCγ levels are dynamic and that in later stages of the disease, its expression is dependent on the degree of motor function in the model. Taken together these results suggest that PKCγ may be a useful marker in the disease to monitor the status of the CST.

The chloride co-transporters, NKCC1 and KCC2, in experimental autoimmune encephalomyelitis (EAE).

Patients with multiple sclerosis (MS) often complain of neuropathic pain. According to the Gate Control Theory of Pain, spinal networks of GABAergic inhibitory interneurons are important in modulating nociceptive inputs from the periphery. Na+-K+-2Cl- co-transporter 1 (NKCC1) and K+-Cl- co-transporter 2 (KCC2) generally dictate the tone of GABA/glycine inhibition by regulating intracellular chloride concentrations. In this study, we investigated the role of NKCC1 and KCC2 in neuropathic pain observed in the animal model, experimental autoimmune encephalomyelitis (EAE), a commonly used model to study the pathophysiology of MS. Quantitative real-time polymerase chain reactions (qRT-PCR) analysis revealed no change in NKCC1 mRNA transcripts in dorsal root ganglia throughout EAE disease course. However, NKCC1 and KCC2 mRNA levels in the dorsal spinal cord were significantly reduced at disease onset and peak only to recover by the chronic time point. Similarly, Western blot data revealed a significant downregulation of NKCC1 and KCC2 in the dorsal spinal cord at disease onset but an upregulation of NKCC1 protein in the dorsal root ganglia at this time point. Treatment with bumetanide, an NKCC inhibitor, had no effect on mechanical hypersensitivity seen in mice with EAE even though it reversed the changes in the levels of NKCC1 and KCC2. We noted that bumetanide treatment, while effective at reversing the changes in monomeric KCC2 levels was ineffective at reversing the changes in oligomeric KCC2 which remained repressed. These results indicate that mechanical hypersensitivity in EAE is not mediated by altered levels of NKCC1.

Antinociceptive Effects of the Antidepressant Phenelzine are Mediated by Context-Dependent Inhibition of Neuronal Responses in the Dorsal Horn

The putative strong anti-nociceptive properties of the antidepressant phenelzine (PLZ) have not been widely explored as a treatment for pain. Antinociceptive effects of PLZ were identified in the formalin model of tonic pain (Mifflin et al., 2016) and in allodynia associated with experimental autoimmune encephalomyelitis, (EAE) a mouse model of multiple sclerosis (Potter et al., 2016). Here, we further clarify the specific types of stimuli and contexts in which PLZ modulates nociceptive sensitivity. Our findings indicate that PLZ selectively inhibits ongoing inflammatory pain while sparing transient reflexive and acute nociception. We also investigated the cellular mechanisms of action of PLZ in the dorsal horn, and as expected of a monoamine-oxidase inhibitor, PLZ increased serotonin (5HT) immunoreactivity. We next used two approaches to test the hypothesis that PLZ inhibits the activation of spinal nociresponsive neurons. First, we evaluated the formalin-evoked protein expression of the immediate early gene, c-fos. PLZ reduced Fos expression in the superficial dorsal horn. Second, we evaluated the effects of PLZ on intracellular calcium responses to superfusion of glutamate (0.3-1.0 mM) in an ex vivo lumbar spinal cord slice preparation. Superfusion with PLZ (100-300 μM) reduced 1 mM glutamate-evoked calcium responses. This was blocked by pretreatment with the 5HT1A-receptor antagonist WAY-100,635, but not the alpha-2 adrenergic antagonist idazoxan. We conclude that PLZ exerts antinociceptive effects through a 5-HT/5HT1AR-dependent inhibition of neuronal responses within nociceptive circuits of the dorsal horn.