Hemodynamic response to intrathecal sildenafil Bombarda G et al. increase in the intracellular cAMP level.12-15 In anesthetized rats, a dose-dependent increase in the MAP following the intrathecal administration of an cGMP analog (8-bromo- cGMP) has been observed. A 8-bromo-cGMP-dependent increase in the MAP suggests that the mechanism underlying this increase involves cGMP.16 As the hemodynamic response caused by sildenafil may involve a lack of cGMP degradation, the second goal of this study was to evaluate the hemodynamic effects of the intrathecal administration of 8-bromo-cGMP in conscious rats and to compare the results with the sildenafil-induced hemodynamic response. Moreover, several studies have proposed that sildenafil, by itself, increases the levels of cAMP.13-15 After taking this idea into account, the final objective of the present study was to examine the cardiovascular response of conscious rats following the intrathecal administration of forskolin, a direct activator of adenylate cyclase that increases the intracellular levels of cAMP, and dibutyryl-cAMP, an analog of cAMP, and to compare the results with the sildenafil-induced response. MATERIAL AND METHODS Animals All experiments were performed on male Wistar rats (320¡30 g) housed individually with free access to food and water. The rats were maintained on a 12512 h light- dark cycle. The experimental protocols used in this research were approved by the Committee of Ethics in Animal Research of the School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (protocol 199/2008). Surgical procedures The rats were anesthetized with tribromoethanol (250 mg/kg, i.p., Sigma Aldrich) and underwent a two- stage surgery. Initially, a polyethylene cannula PE-10 (,15 cm long, total volume 10 mL) filled with heparinized saline (pH 7.4) was inserted through the intervertebral space (L5-L6) into the spinal subarachnoid space in a cranial direction (2 cm). The cannula was then implanted using the method reported by Prado et al.17 with a slight modification developed by Storkson et al.18 Once the procedure was completed, the animals were allowed five days to recover. On the day before the experiment, polyethylene tubing was inserted into the left femoral artery for direct measurement of the arterial pressure. The experiments were performed in conscious rats 24 hours after femoral artery catheterization. Measurement of arterial pressure During the experiments, silence was maintained to avoid undue stress on the animals. The pulsatile arterial pressure was recorded by connecting the arterial catheter to a pressure transducer (P23Gb, Statham, Hato Hey, PR) and was continuously sampled (2 kHz) using an IBM/PC equipped with an analog-to-digital interface (DI-220 Dataq Instruments, Akron, OH). The pulsatile arterial pressure recordings were then analyzed using the CODAS software (Dataq Instruments, Akron, OH) to obtain the MAP and HR values. Experimental protocols The animals received an single intrathecal dose (10 mL) of sildenafil (100 mg, n = 7; 10 mg, n = 6; 1 mg, n = 4; 0.1 mg, CLINICS 2011;66(8):1407-1411 n = 3), 8-bromo-cGMP (0.03 mg, n = 3; 0.47 mg, n = 6; 1.4 mg, n = 7), forskolin (100 mg, n = 7), dibutyryl-cAMP (100 mg, n = 4) or vehicle (1% DMSO, n = 6). The drugs and vehicle (10 mL) were administered via the intrathecal cannula using a Hamilton microsyringe. After drug administra- tion, an additional 10 mL of aCSF was injected to clear the catheter of the drug solution. At the end of the experi- ment, 10 mL of Evan’s blue was injected into the subarachnoid space. The rats were then anesthetized with tribromoethanol (250 mg/kg, i.p.) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffered saline. Finally, a necropsy was performed to confirm the position of the intrathecal catheter and to determine how far the dye spread within the subarach- noid space.17,19 Drugs The drugs used in the current study were sildenafil citrate (Pfizer), 8-bromo-cGMP (8-bromoguanosine 39,59-cyclico- monophosphate sodium salt), dibutyryl-cAMP (N6,29-O- dibutyryladenosine 39,59-cyclic monophosphate sodium salt), and forskolin (Sigma-Aldrich). The vehicle used was DMSO (dimethyl sulfoxide). The components of the aCSF were 127 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3 and 10 mM D- glucose (Sigma-Aldrich). All drugs were dissolved in 1% DMSO immediately before administration, and the pH of the drug solutions was adjusted to 7.4. Statistical analysis The results are expressed as the mean ¡ SEM. Comparisons were performed using one-way and two- way analysis of variance (ANOVA) tests followed by a post- hoc Newman-Keuls test. The statistical differences were considered significant at p,0.05. RESULTS Basal MAP and HR. The baseline MAP and HR in rats that received an intrathecal administration of sildenafil, 8-bromo-cGMP, forskolin, dibutyryl-cAMP, or vehicle (DMSO) were similar (Table 1). Effects of intrathecal administration of sildenafil on the MAP and HR Intrathecal sildenafil administration elicited a dose- dependent increase in the MAP and HR (Figure 1). Although the maximal pressor and tachycardic response induced by 100 and 10 mg of sildenafil were similar, there was a considerable delay in the maximal response with the lower dose. Table1 - Basal hemodynamic data of conscious rats. 1408 DMSO Sildenafil 8-Bromo-cGMP Forskolin Dibutyryl-cAMP MAP (mm Hg) 108¡3 101¡3 104¡2 106¡2 103¡7 HR (bpm) 386¡14 378¡6 381¡6 375¡6 379¡16