Matthew G. Frank

Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation

Activated glial cells (microglia and astroglia) in the spinal cord play a major role in mediating enhanced pain states by releasing proinflammatory cytokines and other substances thought to facilitate pain transmission. In the present study, we report that intrathecal administration of minocycline, a selective inhibitor of microglial cell activation, inhibits low threshold mechanical allodynia, as measured by the von Frey test, in two models of pain facilitation. In a rat model of neuropathic pain induced by sciatic nerve inflammation (sciatic inflammatory neuropathy, SIN), minocycline delayed the induction of allodynia in both acute and persistent paradigms. Moreover, minocycline was able to attenuate established SIN‐induced allodynia 1 day, but not 1 week later, suggesting a limited role of microglial activation in more perseverative pain states. Our data are consistent with a crucial role for microglial cells in initiating, rather than maintaining, enhanced pain responses. In a model of spinal immune activation by intrathecal HIV‐1 gp120, we show that the anti‐allodynic effects of minocycline are associated with decreased microglial activation, attenuated mRNA expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐1β‐converting enzyme, TNF‐α‐converting enzyme, IL‐1 receptor antagonist and IL‐10 in lumbar dorsal spinal cord, and reduced IL‐1β and TNF‐α levels in the CSF. In contrast, no significant effects of minocycline were observed on gp120‐induced IL‐6 and cyclooxygenase‐2 expression in spinal cord or CSF IL‐6 levels. Taken together these data highlight the importance of microglial activation in the development of exaggerated pain states.

A Role for Proinflammatory Cytokines and Fractalkine in Analgesia, Tolerance, and Subsequent Pain Facilitation Induced by Chronic Intrathecal Morphine

The present experiments examined the role of spinal proinflammatory cytokines [interleukin-1β (IL-1)] and chemokines (fractalkine) in acute analgesia and in the development of analgesic tolerance, thermal hyperalgesia, and tactile allodynia in response to chronic intrathecal morphine. Chronic (5 d), but not acute (1 d), intrathecal morphine was associated with a rapid increase in proinflammatory cytokine protein and/or mRNA in dorsal spinal cord and lumbosacral CSF. To determine whether IL-1 release modulates the effects of morphine, intrathecal morphine was coadministered with intrathecal IL-1 receptor antagonist (IL-1ra). This regimen potentiated acute morphine analgesia and inhibited the development of hyperalgesia, allodynia, and analgesic tolerance. Similarly, intrathecal IL-1ra administered after the establishment of morphine tolerance reversed hyperalgesia and prevented the additional development of tolerance and allodynia. Fractalkine also appears to modulate the effects of intrathecal morphine because coadministration of morphine with intrathecal neutralizing antibody against the fractalkine receptor (CX3CR1) potentiated acute morphine analgesia and attenuated the development of tolerance, hyperalgesia, and allodynia. Fractalkine may be exerting these effects via IL-1 because fractalkine (CX3CL1) induced the release of IL-1 from acutely isolated dorsal spinal cord in vitro. Finally, gene therapy with an adenoviral vector encoding for the release of the anti-inflammatory cytokine IL-10 also potentiated acute morphine analgesia and attenuated the development of tolerance, hyperalgesia, and allodynia. Taken together, these results suggest that IL-1 and fractalkine are endogenous regulators of morphine analgesia and are involved in the increases in pain sensitivity that occur after chronic opiates.

mRNA up-regulation of MHC II and pivotal pro-inflammatory genes in normal brain aging

In normal brain aging, CNS resident macrophages exhibit increased expression of major histocompatibility complex (MHC) II expression. However, the transcriptional basis for this observation has not been clarified nor have age-related alterations in pivotal pro-inflammatory genes been characterized. Age-related mRNA alterations in MHC II, MHC II accessory molecules and several pro-inflammatory mediators were measured in older (24 months) and younger (3 months) male F344xBN F1 rats. Real time RT-PCR was utilized to measure steady state mRNA levels in hippocampus. Older as compared to younger animals exhibited increased mRNA levels of MHC II, CD86, CIITA and IFN-gamma. Furthermore, IL-10 and CD200 mRNA, molecules that down-regulate macrophage activation, was decreased in older animals. The present results indicate that normal brain aging is characterized by a shift towards a pro-inflammatory microenvironment in the CNS.

Prior exposure to glucocorticoids sensitizes the neuroinflammatory and peripheral inflammatory responses to E. coli lipopolysaccharide.

Acute and chronic stress has been found to sensitize or prime the neuroinflammatory response to both peripheral and central immunologic challenges. Several studies suggest that stress-induced sensitization of neuroinflammatory processes may be mediated by the glucocorticoid (GC) response to stress. GCs, under some conditions, exhibit pro-inflammatory properties, however whether GCs are sufficient to prime neuroinflammatory responses has not been systematically investigated. In the present investigation, we tested whether acute administration of exogenous GCs would be sufficient to reproduce the stress-induced sensitization of neuroinflammatory responses under a number of different timing relationships between GC administration and immune challenge (lipopolysaccharide; LPS). We demonstrate here that GCs potentiate both the peripheral (liver) and central (hippocampus) pro-inflammatory response (e.g. TNFalpha, IL-1beta, IL-6) to a peripheral immune challenge (LPS) if GCs are administered prior (2 and 24h) to challenge. Prior exposure (24h) to GCs also potentiated the pro-inflammatory response of hippocampal microglia to LPS ex vivo. In contrast, when GCs are administered after (1h) a peripheral immune challenge, GCs suppress the pro-inflammatory response to LPS in both liver and hippocampus. GCs also up-regulated microglial activation markers including Toll-like Receptor 2. The present data suggest that the temporal relationship between GC treatment and immune challenge may be an important factor determining whether GCs exhibit pro- or anti-inflammatory properties.

Time course of hippocampal IL-1 β and memory consolidation impairments in aging rats following peripheral infection

We previously reported that aging F344XBN rats are more vulnerable to disruptions of memory consolidation processes following an injection of Escherichia coli than are young rats. Furthermore, this disruption was specific to hippocampal-dependent memory. In the present study we examined the time course of the proinflammatory cytokine IL-1 beta in young and old rats following a peripheral injection of E. coli. Compared to young rats, aging rats treated with E. coli showed an exaggerated and prolonged up-regulation of IL-1 beta protein in the hippocampus, but not in hypothalamus, parietal cortex, prefrontal cortex, serum or spleen. Aging rats showed greater hippocampal IL-1 beta protein levels than their young counterparts 4h after E. coli, and these levels remained significantly elevated for 8 but not 14 days after E. coli. In a second experiment, aging rats exhibited anterograde memory consolidation impairments 4 and 8 days after an E. coli injection, but not after 14 days. A third experiment revealed that following an E. coli injection, bacterial clearance from the spleen and peritoneum was not impaired in aged rats, suggesting that elevations in hippocampal IL-1 beta were not mediated by impaired clearance in the periphery in aging rats. These data suggest that the exaggerated and prolonged elevation of IL-1 beta, specifically in the hippocampus, may be responsible for hippocampal-dependent memory impairments observed in aging rats following a bacterial infection.

Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain

&NA; Neuropathic pain is a major clinical problem unresolved by available therapeutics. Spinal cord glia play a pivotal role in neuropathic pain, via the release of proinflammatory cytokines. Anti‐inflammatory cytokines, like interleukin‐10 (IL‐10), suppress proinflammatory cytokines. Thus, IL‐10 may provide a means for controlling glial amplification of pain. We recently documented that intrathecal IL‐10 protein resolves neuropathic pain, albeit briefly (˜2–3 h), given its short half‐life. Intrathecal gene therapy using viruses encoding IL‐10 can also resolve neuropathic pain, but for only ˜2 weeks. Here, we report a novel approach that dramatically increases the efficacy of intrathecal IL‐10 gene therapy. Repeated intrathecal delivery of plasmid DNA vectors encoding IL‐10 (pDNA‐IL‐10) abolished neuropathic pain for greater than 40 days. Naked pDNA‐IL‐10 reversed chronic constriction injury (CCI)‐induced allodynia both shortly after nerve injury as well as 2 months later. This supports that spinal proinflammatory cytokines are important in both the initiation and maintenance of neuropathic pain. Importantly, pDNA‐IL‐10 gene therapy reversed mechanical allodynia induced by CCI, returning rats to normal pain responsiveness, without additional analgesia. Together, these data suggest that intrathecal IL‐10 gene therapy may provide a novel approach for prolonged clinical pain control.

Amygdala Regulation of Immediate-Early Gene Expression in the Hippocampus Induced by Contextual Fear Conditioning

The basolateral nuclei of the amygdala (BLA) are thought to modulate memory storage in other brain regions (McGaugh, 2004). We reported that BLA modulates the memory for both an explored context and for contextual fear conditioning. Both of these memories depend on the hippocampus. Here, we examined the hypothesis that the BLA exerts its modulatory effect by regulating the expression of immediate-early genes (IEGs) in the hippocampus. The main findings of these experiments were: (1) Arc activity-regulated cytoskeletal protein (Arc), an immediate-early gene (also termed Arg 3.1) and c-fos mRNA are induced in the hippocampus after a context exposure, or context plus shock experience, but not after an immediate shock; and (2) BLA inactivation with muscimol attenuated the increase in Arc and c-fos mRNA in the hippocampus associated with contextual fear conditioning but did not influence Arc mRNA associated with context exploration. These results support the hypothesis that the amygdala modulates contextual fear memory by regulating expression of IEGs in the hippocampus.

Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: Immunophenotypic and functional characteristics ,

Isolation of microglia from CNS tissue provides a powerful tool to study basic microglia biology and examine the effects of in vivo treatments on microglia immunophenotype and function. Previous microglia isolation methodologies utilized whole brain. However, microglia immunophenotype varies across CNS anatomical loci, thus isolation of microglia from whole brain may obscure regional brain variations in microglia immunophenotype and function. In addition, it is unknown to what extent microglia isolation procedures alter the in situ immunophenotype and function of microglia. The present report details a procedure for the rapid isolation of microglia from discrete CNS anatomical loci and addresses the issue of whether the in situ microglia immunophenotype is significantly altered by the isolation procedure. The present microglia isolation method yielded highly enriched hippocampal microglia, which were devoid of other CNS macrophage subtypes and exhibited attributes reflecting a quiescent phenotype characteristic of microglia observed in situ under non-pathological conditions. Further, isolated microglia exhibited functional responsiveness to immunogenic stimuli ex vivo. The immunophenotypic and functional attributes of isolated microglia suggest that the isolation procedure preserves the in vivo phenotype of microglia, thus providing an experimental method with minimal procedural confounds for examining in vivo treatments on microglia ex vivo.

Intracisternal interleukin-1 receptor antagonist prevents postoperative cognitive decline and neuroinflammatory response in aged rats.

To investigate the role of the pro-inflammatory cytokine interleukin-1β (IL-1β) in postoperative cognitive dysfunction (POCD) in aged rats, we used laparotomy to mimic human abdominal surgery in adult (3 months) and aged (24 months) F344/BN rats. We demonstrated that memory consolidation of the hippocampal-dependent contextual fear-conditioning task is significantly impaired in aged but not young rats 4 d after surgery. Hippocampal-independent auditory-cued fear memory was not disrupted by laparotomy in either age group. The hippocampal-dependent memory impairment was paralleled by elevations of IL-1β in the hippocampus of aged animals 1 and 4 d after surgery. These findings support our substantial line of previous research showing that aged animals are more vulnerable to cognitive decline after a peripheral immune challenge. In addition, we demonstrated that a single intracisternal administration of interleukin-1 receptor antagonist (IL-1RA; 112 μg) at the time of surgery was sufficient to block both the behavioral deficit and the neuroinflammatory response. Injecting the same dose of IL-1RA peripherally failed to have a protective effect. These data provide strong support for the specific role of central, not peripheral, IL-1β in POCD. Furthermore, the long-lasting presence of IL-1RA in the brain (4 d) compared with in the blood (<24 h) underscores the value of intracisternal administration of IL-1RA for therapeutic purposes.

Little Exercise, Big Effects: Reversing Aging and Infection-Induced Memory Deficits, and Underlying Processes

We have previously found that healthy aged rats are more likely to suffer profound memory impairments following a severe bacterial infection than are younger adult rats. Such a peripheral challenge is capable of producing a neuroinflammatory response, and in the aged brain this response is exaggerated and prolonged. Normal aging primes, or sensitizes, microglia, and this appears to be the source of this amplified inflammatory response. Among the outcomes of this exaggerated neuroinflammatory response are impairments in synaptic plasticity and reductions of brain-derived neurotrophic factor (BDNF), both of which have been associated with cognitive impairments. Since it has been shown that physical exercise increases BDNF mRNA in the hippocampus, the present study examined voluntary exercise in 24-month-old F344×BN rats as a neuroprotective therapeutic in our bacterial infection model. Although aged rats ran only an average of 0.7 km per week, this small amount of exercise was sufficient to completely reverse infection-induced impairments in hippocampus-dependent long-term memory compared with sedentary animals. Strikingly, exercise prevented the infection-induced exaggerated neuroinflammatory response and the blunted BDNF mRNA induction seen in the hippocampus of sedentary rats. Moreover, voluntary exercise abrogated age-related microglial sensitization, suggesting a possible mechanism for exercise-induced neuroprotection in aging.