Sarita Garg

Ketamine reduces the cell death following inflammatory pain in newborn rat brain.

Premature infants experience untreated repetitive pain that may alter their brain development. Effects of ketamine and repetitive pain on cellular death and subsequent behavior were studied in neonatal rats. Rat pups were randomized to undisturbed controls (C), 4% formalin injection (F), ketamine alone (K, 5 mg/kg) or formalin plus ketamine (KF) and were assessed for neuroactivation with Fos protein, cellular death with FluoroJade-B, cognition with the radial arm maze, and pain thresholds with the hot-plate. Greater Fos expression and cell death occurred in F vs. C groups in defined brain areas at 1 and 4 h in F compared with other groups. Cell death was accentuated 3.3-fold in cortical areas and 1.6-fold in subcortical areas in the F compared with the C group following repetitive pain and sacrifice 18–20 h later. These effects were ameliorated by ketamine. Compared with the F group, all other groups demonstrated greater exploratory and rearing behaviors and decreased time for bait consumption at 1-h and 3-h intervals. Significantly greater thermal pain latencies occurred in the KF and F groups. Repetitive neonatal pain accentuates neuronal excitation and cell death in developmentally regulated cortical and subcortical areas, which decreases the acquisition of visual-spatial clues, short-term and long-term memory, and increases pain latencies. Ketamine analgesia mitigates most of these effects.

Toll-Like Receptor 2 Modulates the Proinflammatory Milieu in Staphylococcus aureus-Induced Brain Abscess

ABSTRACT Toll-like receptor 2 (TLR2) is a pattern recognition receptor (PRR) that plays an important role in innate immune recognition of conserved structural motifs on a wide array of pathogens, including Staphylococcus aureus. To ascertain the functional significance of TLR2 in the context of central nervous system (CNS) parenchymal infection, we evaluated the pathogenesis of S. aureus-induced experimental brain abscess in TLR2 knockout (KO) and wild-type (WT) mice. The expression of several proinflammatory mediators, including inducible nitric oxide synthase, tumor necrosis factor alpha, and macrophage inflammatory protein-2, was significantly attenuated in brain abscesses of TLR2 KO mice compared to WT mice during the acute phase of infection. Conversely, interleukin-17 (IL-17), a cytokine produced by activated and memory T cells, was significantly elevated in lesions of TLR2 KO mice, suggesting an association between innate and adaptive immunity in brain abscess. Despite these differences, brain abscess severity in TLR2 KO and WT animals was similar, with comparable mortality rates, bacterial titers, and blood-brain barrier permeability, implying a role for alternative PRRs. Expression of the phagocytic PRRs macrophage scavenger receptor type AI/AII and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was increased in brain abscesses of both TLR2 KO and WT mice compared to uninfected animals. However, LOX-1 induction in brain abscesses of TLR2 KO mice was significantly attenuated compared to WT animals, revealing that the TLR2-dependent signal(s) influence LOX-1 expression. Collectively, these findings reveal the complex nature of gram-positive bacterial recognition in the CNS which occurs, in part, through engagement of TLR2 and highlight the importance of receptor redundancy for S. aureus detection in the CNS.

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia.

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro‐inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor‐α and interferon‐γ to induce gap junction coupling in macrophages and microglia, it is possible that pro‐inflammatory mediators released from PGN‐activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus‐derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single‐cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN‐treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation‐induced dye coupling by the gap junction blocker 18‐α‐glycyrrhetinic acid. In contrast to PGN‐activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuroinflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.

Ketamine analgesia for inflammatory pain in neonatal rats: a factorial randomized trial examining long-term effects

BackgroundNeonatal rats exposed to repetitive inflammatory pain have altered behaviors in young adulthood, partly ameliorated by Ketamine analgesia. We examined the relationships between protein expression, neuronal survival and plasticity in the neonatal rat brain, and correlated these changes with adult cognitive behavior.MethodsUsing Western immunoblot techniques, homogenates of cortical tissue were analyzed from neonatal rats 18–20 hours following repeated exposure to 4% formalin injections (F, N = 9), Ketamine (K, 2.5 mg/kg × 2, N = 9), Ketamine prior to formalin (KF, N = 9), or undisturbed controls (C, N = 9). Brain tissues from another cohort of rat pups (F = 11, K = 12, KF = 10, C = 15) were used for cellular staining with Fos immunohistochemistry or FluoroJade-B (FJB), followed by cell counting in eleven cortical and three hippocampal areas. Long-term cognitive testing using a delayed non-match to sample (DNMS) paradigm in the 8-arm radial maze was performed in adult rats receiving the same treatments (F = 20, K = 24, KF = 21, C = 27) in the neonatal period.ResultsGreater cell death occurred in F vs. C, K, KF in parietal and retrosplenial areas, vs. K, KF in piriform, temporal, and occipital areas, vs. C, K in frontal and hindlimb areas. In retrosplenial cortex, less Fos expression occurred in F vs. C, KF. Cell death correlated inversely with Fos expression in piriform, retrosplenial, and occipital areas, but only in F. Cortical expression of glial fibrillary acidic protein (GFAP) was elevated in F, K and KF vs. C. No significant differences occurred in Caspase-3, Bax, and Bcl-2 expression between groups, but cellular changes in cortical areas were significantly correlated with protein expression patterns. Cluster analysis of the frequencies and durations of behaviors grouped them as exploratory, learning, preparatory, consumptive, and foraging behaviors. Neonatal inflammatory pain exposure reduced exploratory behaviors in adult males, learning and preparatory behaviors in females, whereas Ketamine ameliorated these long-term effects.ConclusionNeuroprotective effects of Ketamine attenuate the impaired cognitive behaviors resulting from pain-induced cell death in the cortical and hippocampal fields of neonatal rats. This cell death was not dependent on the apoptosis associated proteins, but was correlated with glial activation.

MyD88 Expression by CNS-Resident Cells is Pivotal for Eliciting Protective Immunity in Brain Abscesses

MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.

Pentoxifylline Enhances the Radioprotective Properties of γ-Tocotrienol: Differential Effects on the Hematopoietic, Gastrointestinal and Vascular Systems

Abstract The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3+PTX before being exposed to 8.5–13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3+PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3+PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3+PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3+PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.

Influence of Sublethal Total-Body Irradiation on Immune Cell Populations in the Intestinal Mucosa

Abstract The intestinal immune system is the largest in the body. This study analyzed changes in intestinal immune cell populations, cytokine protein levels, and transcript profiles after total-body irradiation (TBI) in CD2F1 mice. A single dose of 8.0 Gy γ radiation caused negligible 30-day lethality but induced significant histological damage in jejunal mucosa that was maximal at 3.5 days and that had seemingly recovered by day 21 after irradiation. These changes were accompanied by decreased numbers of mucosal macrophages, neutrophils, and B and T lymphocytes, mostly coinciding with similar reductions in peripheral blood cell counts. Recovery of mucosal macrophages occurred within 1 week, whereas mucosal granulocytes and lymphocytes remained low until 3 weeks after TBI. Maximal suppression of T-helper cell (TH)-related transcripts occurred at 3.5 days, but there was no obvious TH1 or TH2 bias. Genome-wide transcriptional profiling revealed a preponderance of differentially regulated genes involved in cell cycle control, cell death and DNA repair between 4 h and 3.5 days after irradiation. Genes involved in tissue recovery predominated from day 7 onward. We conclude that the intestinal immune system undergoes profound changes after sublethal TBI and that these changes likely contribute to postirradiation pathophysiological manifestations.

Bone Marrow Transplantation Helps Restore the Intestinal Mucosal Barrier after Total Body Irradiation in Mice

Bone marrow transplantation (BMT) substantially improves 10-day survival after total body irradiation (TBI), consistent with an effect on intestinal radiation death. Total body irradiation, in addition to injuring the intestinal epithelium, also perturbs the mucosal immune system, the largest immune system in the body. This study focused on how transplanted bone marrow cells (BMCs) help restore mucosal immune cell populations after sublethal TBI (8.0 Gy). We further evaluated whether transplanted BMCs: (a) home to sites of radiation injury using green fluorescent protein labeled bone marrow; and (b) contribute to restoring the mucosal barrier in vivo. As expected, BMT accelerated recovery of peripheral blood (PB) cells. In the intestine, BMT was associated with significant early recovery of mucosal granulocytes (P = 0.005). Bone marrow transplantation did not affect mucosal macrophages or lymphocyte populations at early time points, but enhanced the recovery of these cells from day 14 onward (P = 0.03). Bone marrow transplantation also attenuated radiation-induced increase of intestinal CXCL1 and restored IL-10 levels (P = 0.001). Most importantly, BMT inhibited the post-radiation increase in intestinal permeability after 10 Gy TBI (P = 0.02) and modulated the expression of tight junction proteins (P = 0.01–0.05). Green fluorescent protein-positive leukocytes were observed both in intestinal tissue and in PB. These findings strongly suggest that BMT, in addition to enhancing general hematopoietic and immune system recovery, helps restore the intestinal immune system and enhances intestinal mucosal barrier function. These findings may be important in the development and understanding of strategies to alleviate or treat intestinal radiation toxicity.

Laser Capture Microdissected Mucosa versus Whole Tissue Specimens for Assessment of Radiation-Induced Dynamic Molecular and Pathway Changes in the Small Intestine

Background The intestinal mucosa is the compartment that sustains the most severe injury in response to radiation and is therefore of primary interest. The use of whole gut extracts for analysis of gene expression may confound important changes in the mucosa. On the other hand, laser capture microdissection (LCM) is hampered by the unstable nature of RNA and by a more complicated collection process. This study assessed, in parallel samples from a validated radiation model, the indications for use of LCM for intestinal gene expression analysis. Methodology/Principal Findings RNA was extracted from mouse whole intestine and from mucosa by LCM at baseline and 4 h, 24 h, and 3.5 d after total body irradiation and subjected to microarray analysis. Among mucosal genes that were altered > = 2-fold, less than 7% were present in the whole gut at 4 and 24 h, and 25% at 3.5 d. As expected, pathway analysis of mucosal LCM samples showed that radiation activated the coagulation system, lymphocyte apoptosis, and tight junction signaling, and caused extensive up-regulation of cell cycle and DNA damage repair pathways. Using similar stringent criteria, regulation of these pathways, with exception of the p53 pathway, was undetectable in the whole gut. Radiation induced a dramatic increase of caspase14 and ectodysplasin A2 receptor (Eda2r), a TNFα receptor, in both types of samples. Conclusions/Significance LCM-isolated mucosal specimens should be used to study cellular injury, cell cycle control, and DNA damage repair pathways. The remarkable increase of caspase14 and Eda2r suggests a novel role for these genes in regulating intestinal radiation injury. Comparative gene expression data from complex tissues should be interpreted with caution.

Procalcitonin as a predictive biomarker for total body irradiation induced bacterial load and lethality in mice

ABSTRACT Sepsis is the leading cause of mortality in intensive care units. Early detection and intervention are critical to prevent death. The acute radiation syndrome is characterized by damage of the gastrointestinal and hematopoietic systems. Translocation of intestinal microflora combined with immune system compromise may lead to septicemia and death. This work examined the utility of procalcitonin, a clinical sepsis biomarker, in a mouse model of radiation toxicity. C57/BL6 mice were exposed to total body irradiation (TBI). Intestinal mucosal permeability was measured in vivo, and liver bacterial load and plasma levels of procalcitonin (PCT), lipopolysaccharide (LPS), and LPS-binding protein were measured at baseline and at 3.5, 7, and 10 days after TBI. The value of early PCT in predicting subsequent lethality was determined by receiver operating characteristic analysis. Four days after TBI, a dose-dependent increase in permeability of the intestinal mucosa was observed, whereas bacterial translocation was present from day 7 onward. There was a high positive correlation between bacterial translocation and all sepsis biomarkers, with PCT exhibiting the strongest correlation. Moreover, plasma PCT levels were elevated already from day 3.5 onward, whereas LPS was elevated from day 7 and LPS-binding protein only 10 days after TBI. Receiver operating characteristic analysis revealed that PCT levels measured 3.5 days after TBI predicted lethality at 10 days. These data demonstrate the value of PCT as an early biomarker in radiation-induced bacteremia for mouse studies and suggest that clinical results from other septic conditions may apply to postradiation septicemia in humans.