Thermal and Cytokine Responses to Endotoxin Challenge During Early Life

Sudden infant death syndrome (SIDS) remains the leading cause of infant mortality beyond the neonatal period. An increase in body temperature as a result of high environmental temperature, over-wrapping of infants and/or infection are associated with SIDS. Endotoxins such as lipopolysaccharide (LPS) and heat stress may perturb cardiorespiratory function and thermoregulation. Although LPS-mediated body temperature and cytokine responses are well-documented in older animals, the capacity of LPS to induce fever and cytokine response in young rats remains unclear. Therefore, we sought to investigate the acute effects of LPS on body temperature and cytokine concentrations in rat pups. Postnatal day 7 rat pups were divided into three groups. Group 1 was administered LPS intraperitoneally (200µg/kg). Group 2 received saline at volume equal to LPS group. Group 3 received no treatment. Pups were placed in custom-made chambers maintained at ambient temperature of 33°C. Body surface temperature was continuously monitored for four hours. Thereafter, the rats were euthanized and serum collected for cytokine analysis. We demonstrate that LPS treatment increased MIP-1α, IL-10, MCP-1, IP-10, fractalkine and TNF-α with no concurrent rise in body surface temperature. Although neonatal rats produced an array of cytokines in response to LPS, there was no evidence of fever.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Quantification of Neu5Ac and Neu5Gc levels in normal and dystrophin-deficient mouse and dog muscles.

<p>Tibialis anterior (TA) and soleus (Sol) muscles were analyzed from 11 wild type (WT) mice, TA and gastrocnemius (GS) muscles were analyzed from 8 mdx mice, and vastus lateralis (VL) and cranial sartorius (CS) muscles were analyzed from 2 golden retriever (GR) and 5 golden retriever muscular dystrophy (GRMD) dogs. Average Neu5Gc as a percentage of total sialic acid is shown. Errors are standard deviation (SD). ***P<0.001, for all dog vs. mouse muscle comparisons.</p

Neu5Gc immunostaining of satellite cells, T lymphocytes and macrophages in GRMD skeletal muscle.

<p>GRMD muscle (cranial sartorius) was triple stained for Neu5Gc (green), Pax7 (A), CD11b (B), CD4 (C), CD8 (D) (all red), and DAPI (blue). Arrows indicated co-staining of Neu5Gc and Pax7 (in A), CD11b (in B) or CD4 (in C). Bar is 100 µm (A) and 50 µm (B–D) for all panels.</p

Neu5Gc expression in GRMD muscle at different ages.

<p>Three different GRMD cases (GRMD1, 2, and 3) were biopsied and muscles stained for Neu5Gc at 3, 6 and 13 months of age. Time-matched images of staining of the cranial sartorius (A) and vastus lateralis (B) muscles are shown. Bar is 50 µm for all panels in A and B.</p

Comparison of CT1 and CT2 immunostaining in DMD and normal human muscle.

<p>CT1 and CT2 were used to immunostain two different DMD (A) and two different normal human (B) muscle biopsies, compared to secondary antibody control. These were compared to time-matched images taken from mdx muscles (C), diaphragm (left panel) or tibialis anterior (right panel), that had been infected with rAAV(rh.74).MCK.GALGT2 for 12 weeks. Bar is 200 µm for all panels in A, B and C.</p

Quantification of CD4−, CD8−, CD11b− and Pax7−stained cells in GR and GRDM muscle and of Neu5Gc co-staining in GRMD muscle.

<p>(A) Cranial sartorius muscle sections from GR and GRMD dogs were stained with markers for satellite cells (Pax7), T lymphocytes (CD4 or CD8) or macrophages (CD11b) and quantified for numbers of cells stained per 40X visual field. (B) The percentage of cells co-stained for Neu5Gc and CD4, CD8, CD11b or Pax7 in GRMD muscles was quantified. Errors are SEM. ***P<0.001, for each GR vs. GRMD comparison in A.</p

Co-staining of CT carbohydrate with macrophages in GRMD muscle.

<p>(A) Single and merged staining for CT2 (green) with CD11b (red, to mark macrophages) and DAPI (blue). Arrow shows co-incident staining for CT2 and CD11b. (B) Single and merged staining for CT2 (green) with Pax7 (red, to mark satellite cells) and DAPI (blue). Arrow shows lack of coincident staining for CT2 and Pax7. (C) Examples of WFA staining (green) with embryonic myosin (red) and DAPI (blue) (two left panels). Examples of control staining for staining shown in A and B using only secondary antibodies with DAPI (two right panels). Bar is 50 µm for all panels in A, B and C.</p

Neu5Gc expression in Golden Retriever cross (GR) and Golden Retriever Muscular Dystrophy (GRMD) dog skeletal muscle relative to mouse.

<p>(A) Neu5Gc-specific affinity-purified chicken IgY was used to stain skeletal muscles from 6 month-old normal Golden Retriever (GR) or Golden Retriever Muscular Dystrophy (GRMD) dogs. An example of staining from a severely affected and a mildly affected GRMD dog are shown. (B) Time-matched images in A were compared to normal mouse skeletal muscle immunostained with the same reagents. Non-immune chicken IgY was used as a control for background staining in A and B. Bar is 200 µm for all panels in A and B.</p