Delayed but Not Early Glucocorticoid Treatment Protects the Host during Experimental Herpes Simplex Virus Encephalitis in Mice

Delayed but Not Early Glucocorticoid Treatment Protects the Host during Experimental Herpes Simplex Virus Encephalitis in Mice

Experimental herpesvirus encephalitis in weanling mice was treated with either cytosine arabinoside or adenine arabinoside to determine the comparative effectiveness of the two compounds on survival and on the concentration of virus in the brain. Most hospitals have a sure area where medical professionals continue to keep these information. Both BVDU and IDU were also effective in suppressing the severity of conjunctivitis, and in this respect, BVDU proved significantly better than IDU. No significant local or systemic toxicity was encountered. In addition, 2′-nor-cGMP was effective orally in preventing HSV-1 orofacial infection and HSV-2 genital infection of mice. Cort was started immediately or 72 h after infection, whereas DEX was administrated 3 days after infection. Results.

You can be skilled in aiding reduce choking incidents, stopping blood movement, or basic splinting of a broken bone. This neurovirulence provoked neuronal death and transcriptional activation of several immune genes. Notably, Toll-like receptors 2, 3, and 9 transcripts were strongly up-regulated. Mean life expectancy was higher in the group of mice that received delayed Cort (56%) than in the mice that received no Cort (44%) or early Cort treatments (13%) (P20% weight loss, isolation, limited movements, or swollen eyes). Brains were removed and post-fixed in a paraformaldehyde solution for 20–24 h, then placed in 10% sucrose diluted in 4% paraformaldehyde-borax buffer at 4°C during several days. All brains were cut in 25-µm coronal sections on dry ice, from the olfactory bulb to the caudal medulla. Can a hundred% Accuracy Be Maintained in Fill Counts for Strong Oral Dosage Varieties?

In a third experiment (real-time polymerase chain reaction [RT-PCR] analysis), mice were infected intranasally with 103 pfu and, 3 days later, received a systemic injection with either saline or DEX as described above. Five mice per group were killed 1, 3, 5, and 7 days after inoculation, and their brains were rapidly removed and snap frozen. The mid-and hindbrain (regions where HSV replicates more aggressively) were extracted and then homogenized in 1 mL of TRIzol reagent (Invitrogen). In situ hybridization. Detection and localization of TNF-α, interferon (IFN)-β, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, and HSV thymidine kinase (TK) transcripts were performed on brain slices using 35S-labeled cRNA probes as described elsewhere [8]. The radioactive antisense cRNA probes were synthesized by incubation of 250 ng of linearized plasmids in 6 mmol/L MgCl2, 40 mmol/L Tris, pH 7.9, 2 mmol/L spermidine, 10 mmol/L NaCl, 10 mmol/L dithiothreitol, 0.2 mmol/L ATP/GTP/CTP, 100 µCi of α-[35S]-UTP (Dupont NEN, NEG 039H), 20 U of RNAsin (Promega), and 10 U of either T7, SP6, or T3 RNA polymerase for 90 min at 37°C [8]. The probes were mixed with a hybridization solution (500 µL of formamide, 60 µL of 5 mol/L NaCl, 10 µL of 1 mol/L Tris, pH 8.0, 2 µLof0.5 mol/L EDTA, pH 8.0, 50 µLof 20×Denhart’s solution, 200 µLof 50% dextran sulfate, 50 µL of 10 mg/mL tRNA, and 10 µLof 1 µol/L dithiothreitol) to reach a final concentration of 105 cpm/mL.

This solution was heated at 60°C for 10 min then spotted on slides containing brain sections. Immunocytochemistry and confocal laser scanning analysis. Brain slices were incubated overnight at 4°C with a polyclonal rabbit anti-HSV I + II (Biogenesis) and a neuronal nuclei (NeuN) antibody (Chemicon International), respectively, diluted 1:100 and 1:800 in KPBS containing 0.4% triton X-100, 1% bovine serum albumin (BSA), and 1% goat serum. After the first incubation, sections were washed and incubated 90 min with the secondary antibody solution containing Alexa Fluor 488-conjugated goat anti-rabbit (1:1000 for HSV-1; Molecular Probes) or Alexa Fluor 594–conjugated goat anti-mouse (1:500 for NeuN; Molecular Probes) in KPBS/0.4% triton X-100/1% BSA. Laser scanning confocal analyses were performed with a BX-61 microscope (Olympus America). Combined immunocytochemistry and in situ hybridization. Immunocytochemistry was combined with in situ hybridization to determine the type of cells expressing IFN-β in the brain of HSV-1-infected mice, by use of previously described methodologies [8].

The presence of the proinflammatory cytokine IFN-β was indicated by the agglomeration of silver grains within cell ramifications (microglia or monocyte/macrophage) labeled with iba1 (Wako Chemicals). Quantitative RT-PCR analysis. Total RNA extraction was performed using the TRIzol method and then digested with deoxyribonuclease (Turbo DNA-free; Ambion). Complementary DNA (cDNA) was generated from 40 ng of total RNA by use of a random primer hexamer in accordance with the protocol for Superscript II (Invitrogen). Equal amounts of cDNA were run in triplicate and amplified in a 15-µL reaction containing 7.5 µLof 2× Universal PCR Master Mix (Applied Biosystems), 10 nmol/L Z-tailed forward primer, 100 nmol/L reverse primer, 100 nmol/L Amplifluor Uniprimer probe (Chemicon), and 2 µL of target DNA. The mixture was incubated at 50°Cfor2min, at 95°C for 4 min, and then cycled at 95°C for 15 s and at 55°C for 40 s 55 times using the Prism 7900 Sequence Detector (Applied Biosystems). Amplification efficiencies were validated and normalized to ribosomal 18S, and amounts of target gene were calculated according to a standard curve.

Delayed but Not Early Glucocorticoid Treatment Protects the Host during Experimental Herpes Simplex Virus Encephalitis in Mice
Primer sequences for immune and viral genes are available on request. Amplicons were detected using the Amplifluor UniPrimer system, in which forward primers contained a 5′Z sequence: ACTGAACCTGACCGTACA. Statistical analysis. Mice were carefully examined twice a day, and obvious sickness signs (>20% weight loss, isolation, limited movement, or swollen eyes) or death were considered as end points for mean life expectancy. Survival curves were estimated by the Kaplan-Meier method and were compared using the log rank test. Quantitative RT-PCR data were analyzed using Student’s t test for independent samples. Statistical analyses were performed with SPSS software (version 11.0; SPSS).

Inflammatory response in the brain of HSV-1-inoculated mice. Clinical signs of infection gradually appeared from day 5 to 8 after intranasal HSV-1 inoculation. The first signs consisted of unilateral ocular swelling. Subsequently, the mice became gradually weaker and lost weight. Finally, some mice had shaking movements and thus were killed immediately. All mice that had clear clinical signs exhibited a similar pattern of HSV TK gene expression (figure 1). At day 7, when the first clinical signs were visible, small-scattered positive cells for TK mRNA were detected in the hindbrain, the midbrain, and a few isolated areas of the rostral brain (figure 1).

In contrast, mice that exhibited severe clinical signs of encephalitis 8–11 days after the inoculation had a robust and widespread hybridization signal for the TK gene across the rostro-caudal brain (figure 1). Rostro-caudal distribution of cells expressing the gene encoding herpes simplex virus (HSV)-1 thymidine kinase (TK) in the brain of infected BALB/c mice. Mice were perfused transcardially with a solution of 4% paraformaldehyde at days 1, 7, and 11 after infection. Rostro-caudal coronal sections from the HSV-1-infected mice show a positive signal on x-ray film (Biomax; exposed at 4°C for 2 days) for TK mRNA in various regions throughout the brain, particularly in the midbrain, pons, and medulla 7 days after inoculating mice with HSV-1. A robust and widespread viral replication was found throughout the entire brain of an infected mouse at day 11 after infection. Antisense cRNA probes were also generated to determine the expression of the different TLRs implicated in infectious microorganism recognition. As shown in figure 2, the pattern of TLR2-positive regions was very similar to the one previously described for the TK gene, which suggests that the capacity of the virus to replicate within specific regions of the brain is linked to the inflammatory response (figure 1 and figure 2).

De novo expression of the TLR2 transcript is a very reliable marker of microglial activation during bacterial infection and brain diseases [11, 17–20]. The cells that contain the virus are nevertheless different, because they are neurons (figure 3). Although TLR2, TLR3, and TLR9 transcripts were strongly up-regulated and widely distributed across the CNS, TLR4 and TLR8 remained almost undetectable. The hybridization signal for TLR6 and TLR7 was present in the pons and medulla but not in the rostral forebrain. Expression of each TLR was therefore very specific. Rostro-caudal distribution of the Toll-like receptor (TLR) family in the brain of herpes simplex virus (HSV)-1-infected BALB/c mice. These coronal sections depict the representative hybridization signal on x-ray film (Biomax; exposed at 4°C for 2–3 days) for the different TLRs at day 7 after HSV inoculation.

Representative examples of the inflammatory response in the brain of mice developing herpes simplex virus (HSV) encephalitis. iba1, ionized calcium binding adapter molecule 1; IFN, interferon; TK, thymidine kinase; TNF-α, tumor necrosis factor-α; NeuN, neuronal nuclei. Several inflammatory genes were also induced in the CNS of mice that exhibited clinical signs of encephalitis (figure 3). This was the case for TNF-α and IFN-β. The expression patterns of these transcripts differed from those previously described, especially for TNF-α, which was found in few regions of the CNS, and the signal intensity was modest, compared with that generated by the TK, TLR2, and TLR3 cRNA probes. Of interest is the highly localized pattern of IFN-expressing cells in the CNS of infected mice (figure 3. The colocalization of these IFN-expressing cells with iba1, along with their morphological characteristics, indicates that these cells may be infiltrating macrophages, but this remains speculative.

In contrast, the TLR2 gene was strongly induced throughout the brain (figure 2). Role of GCs in mice infected with HSV-1. Mice that had free access to Cort in their drinking water before the inoculation with HSV-1 developed clinical signs by as early as 5 days, and their states degenerated rapidly from that point on (figure 4). Conversely, mice had a longer mean life expectancy and developed clinical signs later when the treatment with Cort began 3 days after the inoculation (figure 4), compared with mice that received an early treatment with GCs (P20% weight lost, isolation with no social interaction, limited movement, or swollen eyes) were considered as end points for the mean life expectancy to HSV encephalitis. To be certain that the observed effects were not specific to one drug and one route of administration, a different group of mice received once a day an ip injection with a synthetic form of GCs (DEX) starting on day 3 after inoculation. As shown in figure 5 and table 2, mice that were treated with DEX had a longer life expectancy than the HSV-untreated group (P20% weight lost, isolation with no social interaction, limited movement, or swollen eyes) were considered as end points for the mean life expectancy to HSV encephalitis. Effects of dexamethasone sulfate (DEX) on expression of proinflammatory cytokines (A), interferon (IFN) types I and II and IRF-7 (B), and herpes simplex virus (HSV)-1 replication (thymidine kinase [TK]) mRNA (C).

Specific brain regions (midbrain and hindbrain) of HSV-1-infected mice treated or not with DEX were rapidly removed at day 2±1(10 mice per group) and61± (10 mice per group) after infection and homogenized for RNA extraction. Complementary DNA was synthesized and detected by a standard real-time polymerase chain reaction with specific primers for each gene. Bars show the mean±SE obtained with untreated and DEX-treated groups of infected mice at day 6±1 after infection. *Significantly different (P!

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