ND4 Mice Research Articles

Is Baikiain in Tara Flour a Causative Agent for the Adverse Events Associated with the Recalled Frozen French Lentil & Leek Crumbles Food Product? - A Working Hypothesis.

The French Lentil & Leek Crumbles frozen food product was recently recalled due to reports of gastrointestinal issues. So far, 393 adverse illness complaints and 133 hospitalizations have been reported from consumption of this food, and the tara (Tara spinosa) protein flour ingredient is hypothesized to be responsible. A multipronged approach resulted in identification of (S)-(-)-baikiain in tara as a compound of interest due to its abundance, possible metabolic fate, and close resemblance to irreversible inhibitors of L-pipecolate oxidase. Oral administration of baikiain in ND4 mice showed a statistically significant increase in blood ALT levels and a reduction in liver GSH.

Repeated Vaginal Exposures to the Common Cosmetic and Household Preservative Methylisothiazolinone Induce Persistent, Mast Cell-Dependent Genital Pain in ND4 Mice.

A history of allergies doubles the risk of vulvodynia—a chronic pain condition of unknown etiology often accompanied by increases in numbers of vulvar mast cells. We previously established the biological plausibility of this relationship in mouse models where repeated exposures to the allergens oxazolone or dinitrofluorobenzene on the labiar skin or inside the vaginal canal of ND4 Swiss Webster outbred mice led to persistent tactile sensitivity and local increases in mast cells. In these models, depletion of mast cells alleviated pain. While exposure to cleaning chemicals has been connected to elevated vulvodynia risk, no single agent has been linked to adverse outcomes. We sensitized female mice to methylisothiazolinone (MI)—a biocide preservative ubiquitous in cosmetics and cleaners—dissolved in saline on their flanks, and subsequently challenged them with MI or saline for ten consecutive days in the vaginal canal. MI-challenged mice developed persistent tactile sensitivity, increased vaginal mast cells and eosinophils, and had higher serum Immunoglobulin E. Therapeutic and preventive intra-vaginal administration of Δ9-tetrahydrocannabinol reduced mast cell accumulation and tactile sensitivity. MI is known to cause skin and airway irritation in humans, and here we provide the first pre-clinical evidence that repeated MI exposures can also provoke allergy-driven genital pain.

Pharmacokinetics and Tissue Distribution of Aegeline after Oral Administration in Mice.

Aegeline is claimed to be a biologically active constituent of Aegle marmelos. Preclinical studies have reported possible therapeutic potential for aegeline against obesity and diabetes. In recent years, aegeline has been added to several weight loss products. However, the consumption of aegeline-containing supplements such as OxyELITE Pro and VERSA-1 has been linked to multiple cases of acute and chronic liver failure. This study was carried out to evaluate the pharmacokinetics and tissue distribution of aegeline in ND4 mice. Two doses of aegeline, a human equivalent dose (1×) 30 mg/kg and a 10× dose (300 mg/kg), were orally administered to the mice, and blood and tissue samples were collected over 8 h. The quantitative analysis of plasma and tissue homogenates (liver, kidney, and brain) was done by UHPLC-QTOF to determine aegeline concentrations. The peak plasma level of aegeline was achieved at a Tmax of 0.5 h, indicating its rapid absorption from the gastrointestinal tract. Aegeline was not detected in the plasma at 8 h after oral administration, with a half-life of 1.4 ± 0.01 and 1.3 ± 0.07 h for the 30 and 300 mg/kg doses, respectively. The half-life of aegeline in the liver was 1.2 h and 1.7 h for 30 and 300 mg/kg doses, respectively, with a Tmax of 1.9 h, which indicates relatively fast elimination of aegeline from the liver.

Repeated challenge with the hapten oxazolone provokes persistent vulvar mechanical sensitivity, labiar mast cell accumulation and nerve overgrowth in previously sensitized ND4 mice (HUM1P.270)

Abstract Vulvodynia is a chronic vulvar pain condition affecting up to 8-16% of 18-60 year-old women. Epidemiological studies have associated an increased risk of developing vulvodynia with a history of seasonal and insect allergies. We have previously demonstrated that acute labiar exposure to the hapten allergen oxazolone provokes increased vulvar mechanical sensitivity in ND4 Swiss mice. Here, we show that repeated oxazolone challenge produces persistent vulvar mechanical sensitivity in sensitized mice for at least12 days after 10 allergen exposures. This persistent sensitivity is accompanied by mast cell accumulation and overgrowth of cutaneous neurites both of which persist for at least 14 days after the final allergen exposure. These changes are accompanied by an early and transient increase in mRNAs encoding CADM1 - an adhesion molecule that facilitates nerve-mast cell synapses - in the skin and mRNAs encoding the inflammatory cytokine IL-6 in the spinal cord. Taken together, these neuroimmune biomarkers may serve as potential diagnostic/therapeutic targets for specific allergy-driven subsets of chronic vulvar pain.

Repeated challenge with the hapten oxazolone provokes persistent vulvar mechanical sensitivity and labiar T cell changes in previously sensitized ND4 mice (HYP7P.258)

Abstract Epidemiological evidence suggests a link between environmental allergies and the risk of developing vulvodynia—a chronic provoked vulvar pain condition affecting up to 8-16% of 18-60 year old women. We have previously shown that acute labiar exposure to the hapten allergen oxazolone provokes increased vulvar mechanical sensitivity in ND4 Swiss mice. Here, we show that repeated oxazolone challenge produces persistent vulvar mechanical sensitivity in sensitized mice for at least12 days after final allergen exposure. T cell amplification in the draining iliac lymph nodes resolves within 5 days after final challenge. Persistent sensitivity in the skin is accompanied by sustained changes in skin T cells; CD44+ CD8 and CD4 subsets remain elevated, T cell transcription factors T-bet and FoxP3 continue to be upregulated for up to 17 days after 10 allergen challenges. This is the first characterization of immune infiltrate accompanying sustained allergen-provoked mechanical sensitivity that persists after the resolution of overt inflammatory events following repeated allergen exposures. Our findings can be used to used to elucidate the underlying etiology of allergy-driven subsets of chronic pain conditions.

TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation

Background: Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperalgesia. Methods: We treated ND4 Swiss male mice with intravenous anti-TNF-α antibody or vehicle 30 minutes prior to bilateral, intra-plantar compound 48/80 administration and measured changes in the timing of hind paw withdrawal observed subsequent to mice being placed on a 51oC hotplate. We also assessed changes in tissue swelling, TNF-α gene expression and protein abundance, mast cell degranulation, and neutrophil influx in the hind paw tissue. Findings: We found that TNF-α neutralization significantly blocked thermal hyperalgesia, and reduced early tissue swelling. TNF-α neutralization had no significant effect on mast cell degranulation or neutrophil influx into the tissue, however. Moreover, no changes in TNF-α protein or mRNA levels were detected within 3 hours of administration of compound 48/80. Interpretation: The neutralizing antibodies likely target pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Pre-formed TNF-α, released upon degranulation, has immediate effects on nociceptive signaling prior to the induction of neutrophil influx. These early effects on nociceptors are abrogated by TNF-α blockade, resulting in compromised nociceptive withdrawal responses to acute, harmful environmental stimuli.

TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation

Background: Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperalgesia. Methods: We treated ND4 Swiss male mice with intravenous anti-TNF-α antibody or vehicle 30 minutes prior to bilateral, intra-plantar compound 48/80 administration and measured changes in the timing of hind paw withdrawal observed subsequent to mice being placed on a 51oC hotplate. We also assessed changes in tissue swelling, TNF-α gene expression and protein abundance, mast cell degranulation, and neutrophil influx in the hind paw tissue. Findings: We found that TNF-α neutralization significantly blocked thermal hyperalgesia, and reduced early tissue swelling. TNF-α neutralization had no significant effect on mast cell degranulation or neutrophil influx into the tissue, however. Moreover, no changes in TNF-α protein or mRNA levels were detected within 3 hours of administration of compound 48/80. Interpretation: The neutralizing antibodies likely target pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Pre-formed TNF-α, released upon degranulation, has immediate effects on nociceptive signaling prior to the induction of neutrophil influx. These direct effects on nociceptors are abrogated by TNF-α blockade resulting in compromised nociceptive withdrawal responses to acute, harmful environmental stimuli.

Deimination restores inner retinal visual function in murine demyelinating disease

Progressive loss of visual function frequently accompanies demyelinating diseases such as multiple sclerosis (MS) and is hypothesized to be the result of damage to the axons and soma of neurons. Here, we show that dendritic impairment is also involved in these diseases. Deimination, a posttranslational modification, was reduced in the retinal ganglion cell layer of MS patients and in a transgenic mouse model of MS (ND4 mice). Reduced deimination accompanied a decrease in inner retinal function in ND4 mice, indicating loss of vision. Local restoration of deimination dramatically improved retinal function and elongation of neurites in isolated neurons. Further, neurite length was decreased by downregulation of deimination or siRNA knockdown of the export-binding protein REF, a primary target for deimination in these cells. REF localized to dendrites and bound selective mRNAs and translation machinery to promote protein synthesis. Thus, protein deimination and dendritic outgrowth play key roles in visual function and may be a general feature of demyelinating diseases.

The role of deimination in ATP5b mRNA transport in a transgenic mouse model of multiple sclerosis

Deimination refers to conversion of protein-bound arginine into citrulline. An mRNA carrier, RNA binding export factor (REF), present on mitochondria undergoes loss of deimination with impaired ATP5b mRNA transport in ND4 mice (model of multiple sclerosis) compared with the controls. We present evidence of (1) reduced ATP5b mRNA binding strength of non-deiminated REF compared with deiminated REF, (2) impaired ATP5b mRNA transport in ND4 mice and (3) reduced mitochondrial ATP synthase activity on inhibition of deimination in PC12 cells. Impaired deimination of REF and defect in mitochondrial mRNA transport are critical factors in mitochondrial dysfunction in ND4 mice.

Evaluation of a Transgenic Mouse Model of Multiple Sclerosis with Noninvasive Methods

To evaluate the ND4 transgenic mouse model of multiple sclerosis using noninvasive methods. Assessment of neurologic/behavioral abnormalities was made using pattern electroretinogram (PERG), magnetic resonance imaging (MRI), optic coherence tomography (OCT), and end point histologic analysis. Electrophysiologic (PERG) recordings demonstrated functional deficits in vision commensurate with neurologic/behavioral abnormalities. In ND4 mice, the authors found PERG abnormalities preceded neurologic/gait abnormalities. MRI demonstrated subtle structural changes that progressed over time in correlation with behavioral abnormalities. The ND4 mouse model has been evaluated using well-defined parameters of noninvasive methods (PERG, MRI, and OCT), enabling objective identification of functional and structural deficits and their correlation with neurologic/gait abnormality.

Mouse Genotype Alters Palatal Fusion Response to Nicotine

OBJECTIVEOur previous work demonstrated that nicotine inhibited palatal fusion in vitro in a dose dependent manner. We asked if the mouse genotype would affect palatal fusion when exposed to nicotine. The objective of this study is focused on understanding the risk of maternal smoking as a factor for facial cleft defects.METHODSPalatal shelves from mouse strains (CD1, ND4, C57and 129S) at age E13.5 were cultured in serum free media (control), and nicotine (0.6 mM and 6 mM). Palates were harvested at 24 hours, 48 hrs and 72 hrs. Tissue was fixed in 4%PFA for paraffin embedding and H&E sections. In vivo study was done using pumps with nicotine in the four different mouse strains. Embryos mice were harvested at E15.5. Palatal tissue was harvested and fixed in 4% PFA for paraffin embedding and H&E sections.RESULTSThe different mouse strains responded to the nicotine in different degrees. All palates exposed to nicotine failed to fuse. CD1 and ND4 strains were consistent in the palatal fusion pattern within litters. C57 and 129S had variable palate fusion. Some of the embryos had fused palates at embryonic day 13.5, while other embryos had undeveloped palatal shelves. The organ cultured palates from CD1 and ND4 mice treated with 0.6mM nicotine fused, while palatal shelves from C57 and 129S didn't fuse. The results in the in vivo experiment showed that the patterns in fusion occurred differently anterior to posterior especially in CD1 mice, the shelves were not fused in the posterior region.CONCLUSIONThe genetic background of mice influenced nicotine responses in palatal tissues.Grant Funding Source: March of Dimes

Hypoxic Preconditioning-Induced Cerebral Ischemic Tolerance

The importance of bioactive lipid signaling under physiological and pathophysiological conditions is progressively becoming recognized. The disparate distribution of sphingosine kinase (SphK) isoform activity in normal and ischemic brain, particularly the large excess of SphK2 in cerebral microvascular endothelial cells, suggests potentially unique cell- and region-specific signaling by its product sphingosine-1-phosphate. The present study sought to test the isoform-specific role of SphK as a trigger of hypoxic preconditioning (HPC)-induced ischemic tolerance. Temporal changes in microvascular SphK activity and expression were measured after HPC. The SphK inhibitor dimethylsphingosine or sphingosine analog FTY720 was administered to adult male Swiss-Webster ND4 mice before HPC. Two days later, mice underwent a 60-minute transient middle cerebral artery occlusion and at 24 hours of reperfusion, infarct volume, neurological deficit, and hemispheric edema were measured. HPC rapidly increased microvascular SphK2 protein expression (1.7+/-0.2-fold) and activity (2.5+/-0.6-fold), peaking at 2 hours, whereas SphK1 was unchanged. SphK inhibition during HPC abrogated reductions in infarct volume, neurological deficit, and ipsilateral edema in HPC-treated mice. FTY720 given 48 hours before stroke also promoted ischemic tolerance; when combined with HPC, even greater (and dimethylsphingosine-reversible) protection was noted. These findings indicate hypoxia-sensitive increases in SphK2 activity may serve as a proximal trigger that ultimately leads to sphingosine-1-phosphate-mediated alterations in gene expression that promote the ischemia-tolerant phenotype. Thus, components of this bioactive lipid signaling pathway may be suitable therapeutic targets for protecting the neurovascular unit in stroke.

Murine Genotype Alters Palatal Tissue Response to Nicotine

Our previous work demonstrated that nicotine inhibited palatal fusion in vitro in a dose dependent manner. We asked if the mouse genotype would be a factor in nicotine teratology.METHODSPalatal shelves from mouse strains (CD1, ND4, C56 and 129S) E13.5 were cultured in serum free media (control), and nicotine (0.6 mM and 6 mM). Palates were harvested at 24 hours, 48 hrs and 72 hrs and fixed in 4%PFA for paraffin embedding and H&E sections.RESULTSCD1 and ND4 strains were consistent in the palatal fusion pattern within litters. The shelves in all the embryos are not fused at a specific age (E 13.5).C56 and 129Shad variable palate fusion. Some of the embryos had fused palates at embryonic day 13.5, while other embryos had undeveloped palatal shelves. The tissue cultured palates from CD1 and ND4 mice treated with 0.6mM nicotine fused, while palate treated with 6 mM nicotine retained epithelial seam. Palates from C56 mice had an epithelial seam using low and high doses of nicotine. 129S palate shelves 6mM nicotine have two layers of MEE in the midline (non fusion).CONCLUSIONSThe genetic background of mice influenced nicotine responses in palatal tissues.Grant Funding Sources

Microvascular Sphingosine Kinase Isoforms in Cerebral Hypoxic Preconditioning

Hypoxic preconditioning (HPC) provides robust neuroprotection against ischemia‐induced neurovascular dysfunction and cell death following stroke and may depend on augmentation of Akt and eNOS signaling pathways. Studies of preconditioning in myocardium reveal sphingosine kinase (SphK) activity could be involved. To test the hypothesis that cerebral HPC may also require SphK, we preconditioned Swiss‐Webster ND4 mice for 4 h at 8% oxygen and measured SphK1 and SphK2 expression in cortical microvessel lysates by immunoblotting. Although no change in SphK1 level was detected, SphK2 increased 2‐fold versus sham (P<0.05) 2 h post‐HPC. No change was seen prior to 2h and SphK2 returned toward baseline after 2h. The SphK2 product S1P induces Akt and eNOS phosphorylation, suggesting that SphK2 upregulation may contribute to ischemic protection promoted by HPC. SphK activity and knockdown experiments to identify the role of SphK2 in HPC signaling by Akt and eNOS are underway in a cerebral endothelial cell culture model of HPC. These studies indicate SphK2 may be important in establishing a vasculo‐protective phenotype in the cerebral microcirculation in response to HPC. Supported by NIH grant HL79278.

Long-Term Tolerance to Retinal Ischemia by Repetitive Hypoxic Preconditioning: Role of HIF-1α and Heme Oxygenase-1

To determine whether the duration of ischemic tolerance in the retina could be extended by repetitive presentations of the preconditioning stimulus and to begin to elucidate the mechanistic underpinnings of the resultant novel phenotype. Adult male Swiss-Webster ND4 mice were repeatedly preconditioned with systemic hypoxia (RHP) over 12 days; 4 weeks later, the mice were subjected to 30 minutes of unilateral retinal ischemia. Protection was quantified morphologically and functionally 1 week after ischemia by histologic analyses and scotopic electroretinography, respectively. Temporal expression patterns of hypoxia-inducible factor (HIF)-1alpha and heme oxygenase (HO)-1 were measured in response to RHP and after retinal ischemia by immunoblot analysis and immunohistochemistry. Morphologic and functional protection against ischemia-induced reductions in retinal layer thicknesses and layer cell counts, and a- and b-wave amplitudes, was documented for at least 4 weeks after RHP. There was no evidence of tissue injury or dysfunction by RHP alone. Temporally associated with this period of long-term tolerance (LTT) to retinal ischemia were sustained increases in retinal levels of HIF-1alpha and HO-1 protein lasting at least 1 and 4 weeks, respectively, after the last RHP stimulus. A novel form of sustained retinal ischemic tolerance is described, wherein endogenous adaptive responses triggered by repeated hypoxia afford protection against injury many weeks after the preconditioning stimulus. HIF-1alpha-mediated, long-lasting increases in retinal HO-1 expression may contribute to the LTT phenotype. Further elucidation of the genetic and molecular basis of such adaptive plasticity could provide therapeutic targets for preventing and/or treating a variety of ischemic retinopathies.

Partitioning of myelin basic protein into membrane microdomains in a spontaneously demyelinating mouse model for multiple sclerosisThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

We have characterized the lipid rafts in myelin from a spontaneously demyelinating mouse line (ND4), and from control mice (CD1 background), as a function of age and severity of disease. Myelin was isolated from the brains of CD1 and ND4 mice at various ages, and cold lysed with 1.5% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate). The lysate was separated by low-speed centrifugation into supernatant and pellet fractions, which were characterized by Western blotting for myelin basic protein (MBP) isoforms and their post-translationally modified variants. We found that, with maturation and with disease progression, there was a specific redistribution of the 14-21.5 kDa MBP isoforms (classic exon-II-containing vs exon-II-lacking) and phosphorylated forms into the supernatant and pellet. Further fractionation of the supernatant to yield detergent-resistant membranes (DRMs), representing coalesced lipid rafts, showed these to be highly enriched in exon-II-lacking MBP isoforms, and deficient in methylated MBP variants, in mice of both genotypes. The DRMs from the ND4 mice appeared to be enriched in MBP phosphorylated by MAP kinase at Thr95 (murine 18.5 kDa numbering). These studies indicate that different splice isoforms and post-translationally modified charge variants of MBP are targeted to different microdomains in the myelin membrane, implying multifunctionality of this protein family in myelin maintenance.

Monoclonal antibodies binding to the cell surface of Listeria monocytogenes serotype 4b

Serotype 4b strains of the food-borne pathogen Listeria monocytogenes are responsible for a large portion of sporadic listeric infections and all major food-borne listeriosis outbreaks in humans. Hybridomas were produced from three fusions with lymphocytes of ND4 mice immunized either with the insoluble antigens of L. monocytogenes serotype 4b or with formalin-killed bacterial cells and screened for monoclonal antibodies (mAbs) reactive to L. monocytogenes serotype 4b. A set of 35 mAbs was identified by ELISA as having reactivity with both the insoluble antigen fraction and the whole-cell antigens. Thirteen of these mAbs belonged to immunoglobulin subclass G1 (IgG1), fifteen were IgG2a and seven mAbs were IgM. Only 20 out of the 35 mAbs were capable of detecting protein bands of various sizes ranging from 20 to 88 kDa in Western blots. Two of these mAbs, M2365 and M2367, were capable of binding to cell-surface antigens of live L. monocytogenes serotype 4b, as demonstrated by immunofluorescence microscopy and immunogold transmission electron microscopy. Immunofluorescence microscopy showed that M2365 and M2367 failed to bind to the cell surfaces of Escherichia coli O157 : H7, Salmonella enterica (serotype Typhimurium DT104) or Campylobacter jejuni. Evaluation of the cross-reactions of all 35 mAbs with whole-cell antigens of E. coli O157 : H7, S. Typhimurium, C. jejuni and Listeria innocua by ELISA indicated that the majority of the mAbs, including M2365 and M2367, did not cross-react with E. coli O157 : H7, S. Typhimurium or C. jejuni and showed no or a very weak reaction with L. innocua. Furthermore, M2365 and M2367 showed no reaction with whole-cell antigens derived from L. monocytogenes serotypes 1/2a, 1/2b and 3a, and from Listeria grayi, Listeria ivanovii and Listeria seeligeri, in an ELISA. Collectively, these data suggest that M2365 and M2367 have potential use in the development of immunological methods of laboratory diagnosis for L. monocytogenes serotype 4b in clinical or food samples.

Generation of Antibodies against Bovine Recombinant Prion Protein in Various Strains of Mice

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, belong to a group of neurodegenerative disorders affecting humans and animals. To date, definite diagnosis of prion disease can only be made by analysis of tissue samples for the presence of protease-resistant misfolded prion protein (PrP(Sc)). Monoclonal antibodies (MAbs) to the prion protein provide valuable tools for TSE diagnosis, as well as for basic research on these diseases. In this communication, the development of antibodies against recombinant bovine prion protein (brecPrP) in four strains of mice (BALB/c, ND4, SJL, and NZB/NZW F(1)) is described. Immunization of autoimmunity-prone NZB/NZW F(1) and SJL mice with brecPrP was applied to overcome self-tolerance against the prion protein. ND4 and SJL mice did not develop an immune response to brecPrP. BALB/c mice produced antibody titers of 1:1,000 to 1:1,500 in an enzyme-linked immunosorbent assay (ELISA), while NZB/NZW F(1) mice responded with titers of 1:7,000 to 1:11,000. A panel of 71 anti-brecPrP MAbs recognizing continuous and discontinuous epitopes was established from BALB/c and NZB/NZW F(1) mice. Seven anti-brecPrP MAbs reacted with both the cellular form of PrP and protease K-resistant PrP(Sc) from sheep brain in Western blot assays. The epitope specificity of these MAbs was determined, and applicability to immunohistochemical detection of prions was studied. The MAbs generated will be useful tools in the development of TSE immunochemical diagnosis and for research. This is the first report of the development of anti-PrP MAbs by use of autoimmune NZB/NZW F(1) mice as an alternative approach for the generation of PrP-specific MAbs.

Early antibiotic administration but not antibody therapy directed against IL-6 improves survival in septic mice predicted to die on basis of high IL-6 levels

Elevated interleukin (IL)-6 levels correlate with increased mortality following sepsis. IL-6 levels >14,000 pg/ml drawn 6 h after cecal ligation and puncture (CLP) are associated with 100% mortality in ND4 mice, even if antibiotic therapy is initiated 12 h after septic insult. Our first aim was to see whether earlier institution of antibiotic therapy could improve overall survival in septic mice and rescue the subset of animals predicted to die on the basis of high IL-6 levels. Mice (n = 184) were subjected to CLP, had IL-6 levels drawn 6 h later, and then were randomized to receive imipenem, a broad spectrum antimicrobial agent, beginning 6 or 12 h postoperatively. Overall 1-wk survival improved from 25.5 to 35.9% with earlier administration of antibiotics (P < 0.05). In mice with IL-6 levels >14,000 pg/ml, 25% survived if imipenem was started at 6 h, whereas none survived if antibiotics were started later (P < 0.05). On the basis of these results, we examined whether targeted antibody therapy could improve survival in mice with elevated IL-6 levels. A different cohort of mice (n = 54) had blood drawn 6 h after CLP, and then they were randomized to receive either monoclonal anti-IL-6 IgG or irrelevant rat IgG. Anti-IL-6 antibody failed to improve either overall survival or outcome in mice with IL-6 levels >14,000 pg/ml. These results demonstrate that earlier systemic therapy can improve outcome in a subset of mice predicted to die in sepsis, but we are unable to demonstrate any benefit in similar animals using targeted therapy directed at IL-6.

AGE DISPROPORTIONATELY INCREASES SEPSIS-INDUCED APOPTOSIS IN THE SPLEEN AND GUT EPITHELIUM

Both aging and sepsis independently increase splenic and gut epithelial apoptosis. Sepsis-induced apoptosis in either cell type is also associated with increased mortality in young mice. We sought to determine whether age alters sepsis-induced splenic and gut epithelial cell death. Young (2 months) and aged (22 months) male ND4 mice were subjected to either single-puncture cecal ligation and puncture (CLP) with a 23-gauge needle or sham laparotomy. Apoptosis was assessed 24 hours later in the spleen and gut epithelium by active caspase 3 and hematoxylin and eosin staining. Aged septic mice had increased splenic apoptosis compared with either young septic animals or aged sham animals (15 vs. 7 vs. 5 apoptotic cells/high-powered field, P < 0.05). Similarly, aged septic animals had an elevation in gut epithelial cell death compared with either young septic or aged sham mice (33 vs. 16 vs. 6 apoptotic cells/100 contiguous crypts, P < 0.05). Elevated intestinal cell death was not associated with changes in either gut proliferation or cell division. To verify that the increase in splenic apoptosis seen in septic aged animals was not strain specific, double-puncture CLP with a 25-gauge needle or sham laparotomy was performed on young (4 months) or aged (24 months) C57BL/6 male mice. Similar to results seen in outbred animals, aged septic animals in this inbred strain had increased splenic apoptosis compared with either young septic animals or aged sham animals (23 vs. 7 vs. 4 apoptotic cells/ high powered field, P < 0.05). These results indicate that although infection and aging each independently cause an increase in splenic and gut epithelial apoptosis, their combination leads to a disproportionate increase in cell death in these rapidly dividing cell populations,and potentially plays a role in the marked increase in mortality seen with aging in sepsis.