Rats were anesthetized with isoflurane and then injected into the 3V with either 25 µL of the control Ad-shβGal-EGFP stock solution (108 IFU/mL) or 25 µL of the Ad-shGK-EGFP stock solution (2 × 108 IFU/mL) for the experimental group at 2.5 µL/min. We were not able to further concentrate the adenovirus in order to inject it in a lower volume. Brains were collected for immunohistochemistry at 18, 24 and 48 h. The rat brains were fixed in 4% paraformaldehyde (PFA) by immersion for 48 h. After fixation, thick frontal sections of the hypothalamus (40 µm) were cut with a cryostat and subsequently processed free-floating. Tissues were immunostained with mouse anti-vimentin (1:200, DAKO, Carpinteria, CA, USA), rabbit anti-glial fibrillary acidic protein (GFAP; 1:200, DAKO) and rabbit anti-NeuN (1:1000, Abcam, Cambridge, MA, USA) antibodies diluted in Tris-HCl buffer (pH 7.8) containing 8.4 mM sodium phosphate, 3.5 mM potassium phosphate, 120 mM sodium chloride, and 1% bovine serum albumin. Sections were incubated with the primary antibodies overnight at room temperature in a humid chamber. After extensive washing, sections were incubated for 2 h at room temperature with Cy2-,Cy3- or Cy5-labeled secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA). These samples were counterstained with the DNA stain TOPRO-3 (1:1000; Invitrogen). The slides were analyzed using confocal laser microscopy (LSM 700, Zeiss). Colocalization of different markers was assessed by measuring the Pearson’s correlation coefficient, Rr. An Rr value of ‘1’ indicates complete colocalization, and a Rr value of ‘0’ indicates no specific colocalization.