Dbcamp

An excitotoxic lesion was created in the hippocampi of 4-months-old male BALB/c mice by injecting 1 μl (0.25 μg) of kainic acid (Sigma–Aldrich, St. Louis, MO, USA) into the lateral ventricle of both sides. Lesioned mice (L) were divided into L+dBcAMP and L+phosphate-buffered saline (PBS) groups. Sham surgery (S) was done by the injection of 1 μl of sterile saline into the lateral ventricles of both sides. The sham surgery mice were divided into S+dBcAMP and S+PBS groups. The mice in the L+dBcAMP and S+dBcAMP groups were treated with dBcAMP (Sigma–Aldrich, St. Louis, MO, USA) daily through the intraperitoneal route at a dose of 50 mg/kg for 1 week (i.p., 50 mg/kg/day) from the day of lesion, whereas the mice in the L+PBS and S+PBS groups were treated with PBS. A total of 48 mice (n = 12/group) were used in the study (Table 1). The mice in all groups were subjected to the water maze and the passive avoidance tests at the end of the 4th week. Morphological studies using Cresyl violet staining, NeuN immunostaining (for neuron), and DCX immunostaining (for neurogenesis) of brain sections were done. Hippocampal tissues were further analyzed for doublecortin content by Western blot.

A stab wound was done on the cerebral cortex of 2 months old BALB/c male mice as described earlier (Abd-El-Basset et al., 1989 (link)). Briefly, mice were anaesthetized with ketamine (40mg/kg)-xylocane (5mg/kg) mixture (Sigma Chemicals, St. Louis, USA), a stab-wound was made by inserting 21-gauge sterile needle into right frontal cerebral cortex (3 mm depth from skull surface) and 2 mm to the right side of the midline. Animals were divided into two sub-groups: dBcAMP group: mice in this group were treated with dBcAMP (ip, 50 mg/kg, Sigma Chemicals, St. Louis, USA) every day for 3, 5, and 7 days; and control group: mice in this group were treated with PBS for 3, 5, and 7 days. Mice in all groups were euthanized with CO2, perfused with cold PBS on 3rd, 5th, 7th post injury days and fresh tissues around the wound (5mm³) was collected for analysis of GFAP content and BDNF level by Western blot and ELISA methods respectively. Additional mice in each group were perfused with saline followed by 4% paraformaldehyde for morphological study by Cresyl violet staining, histochemical staining (Flourojade-B) for neurodegeneration (Schmued and Hopkins, 2000 (link)) and immunostaining for astrocytes (GFAP) and microglia (Iba1). Six mice were used in each sub-group. Three independent experiments were done. A total of 108 mice were used for this experiment (Table 1).

Yeast cells of C. albicans (2.5 × 10³/well) were plated onto 96-well plates containing M199 and treated with 5 μg/mL (based on sterol content) of C. albicans 90028 EVs preincubated with the cAMP donor db-cAMP (10 mM) (Sigma-Aldrich) for 1 h. Alternatively, yeasts were treated with C. albicans 90028 EVs for 1 h, and then db-cAMP (10 mM) was added to the wells. Cell morphology was analyzed using an Observer Z1 microscope (Carl Zeiss International, Germany) after 4, 8, and 24 h.