Table 1.

Animal studies investigating PACAP and the blood-brain barrier

Study	Aims	Method	Outcome	Comments
Banks, 1993 [10]	Investigate BBB permeability (influx) for PACAP in the mouse. Efflux from brain to blood was also investigated.	I125-labelled PACAP was injected in the jugular vein and blood samples from the contralateral carotid artery after 0.5 to 5 min. Decapitation of the animal immediately after last blood sample. Serum and brain tissue was counted in a gamma counter to determine influx constant Ki. For efflux, I125-labelled PACAP was injected in the left ventricle and the brain was removed 2 to 20 min after. Whole brain was counted in a gamma counter.	Influx, Ki for PACAP27 was 2.13 uL/g/min and for 2.86 uL/g/min for PACAP38. Efflux. T1/2 for PACAP27 was 11.8 min and 22.3 min for PACAP38. Unlabelled PACAP38 inhibited influx of labelled PACAP38, which was not true for PACAP27. Unlabelled PACAPs inhibited efflux for their respective counterparts.	0.053% of the iv injected PACAP38 was found in the brain after 5 min or 0.118%iv/g. PTS-6 was suggested to be responsible for active saturable transport across the BBB.
Banks, 1998 [11]	Investigate effect of spinal cord injury on the BBB permeability for PACAP38 and albumin in the mouse.	See Banks, 1993.	Control (healthy) mouse Ki for PACAP38 was 2.42 μL/g/min. In the injuried mouse BBB permeability decreased immediately after injury but increased from day 7 after injury.	I-PACAP was inhibited by unlabeled PACAP. BBB permeability for PACAP was unrelated to the permeability to albumin.
Mizushima, 1999 [12]	Investigate BSCB and BBB permeability for PACAP38 after experimentally induced cardiac arrest in the mouse.	See Banks, 1993. I131-PACAP was used instead of I125-PACAP.	Reversible increase in BSCB permeability for PACAP38 but no BBB change after cardiac arrest.	The actual BBB permeability values were not reported.
Mizushima, 2000 [13]	Investigate BSCB and BBB permeability for PACAP38 after experimentally induced cardiac arrest in the mouse.	See Banks, 1993. I131-PACAP was used instead of I125-PACAP.	Reversible increase in BSCB permeability for PACAP38 but no BBB change after cardiac arrest.	The actual BBB permeability values were not reported.
Somogyvári-Vigh, 2000 [19]	Investigate BBB permeability for PACAP38 and albumin after experimental MCA occlusion in the rat.	See Banks, 1993. The penile vein was used for i.v. injection of PACAP instead of the jugular vein.	Ki was 6.5 μL/g/min before and 7.95 μL/g/min after MCA occlusion.	Increased permeability for PACAP38 but not albumin up to 4 h after and normal at 48 h.
Nonaka, 2002 [14]	Investigate BBB permeability for PACAP38 between young and aged mice.	See Banks, 1993. I131-PACAP was used instead of I125-PACAP.	Young ICR mice: Ki was 7.95 uL/g/min for whole brain. Lowest Ki in the frontal cortex: 3.81 uL/g/min and highest Ki in the hypothalamus: 54.87 uL/g/min and hippocampus: 24.53 uL/g/min. Young SAMP8 mice: Ki was 5.31 uL/g/min for whole brain. Lowest Ki in the thalamus: 3.06 uL/g/min and highest Ki in the hippocampus: 16.96 uL/g/min and hypothalamus: 16.24 uL/g/min. Aged SAMP8 mice: Ki was 2.51 uL/g/min for whole brain. Lowest Ki in the frontal cortex: 2.11 uL/g/min and highest Ki in the hippocampus and hypothalamus: 7.79 uL/g/min.	ANOVA showed difference between young and aged mice. The whole brain difference was only significant between young ICR and aged SAMP8 mice.
Nonaka, 2005 [15]	Investigate BBB permeability (influx) for PACAP38 after intraperitoneal LPS injection in the mouse. Efflux from brain to blood was also investigated.	See Banks, 1993. I131-PACAP was used instead of I125-PACAP.	PACAP transport rate from blood to brain was not altered by LPS. Efflux. T1/2 for PACAP38 was unchanged 15.6 min after LPS.	Albumin permeability increased after LPS suggesting BBB disruption by the LPS.
Dogrukol-Ak, 2009 [16]	Investigate BBB permeability (efflux) for PACAP27 and PACAP38 in the mouse.	The animal was anesthezised and MCA occlusion was induced. Subsequently, I131-labelled PACAPs with and without a selective antisense targeting the efflux component was injected in the jugular vein. 24 h after MCA occlusion the animal was decapitated and the brain tissue was counted in a gamma counter to determine accumulation of PACAPs in the brain.	PACAP27 but not PACAP38 accumulated in the brain after selective inhibition of the beta-F1 ATPase (efflux component of the PTS-6).	PACAP38 and PACAP27 transport from brain to blood may be via two different mechanisms.
Nonaka, 2012 [17]	Investigate uptake of PACAP38 in the brain after intranasal administration in the mouse.	Intranasal administration of I131 labelled and unlabeled PACAP38 and blood sampling from the carotid artery. Decapitation 5–120 min after administration. Serum and brain tissue was counted in a gamma counter to determine	I-PACAP38 uptake occurred in all examined brain regions with the highest uptake in the occipital cortex and striatum (2–4%inj/g). Concomitant administration of unlabeled PACAP38 increased I-PACAP38 in the brain significantly (1000-fold).	Increased amount of I-PACAP38 after concomitant PACAP administration suggests competitive inhibition of the efflux component.
Yu, 2012 [18]	Investigate BBB permeability for PACAP38 with and without combining it with TAT in the mouse.	Recombinant TAT-PACAP38 and PACAP38 were both labeled with FITC and injected i.p. 6 h after injection the animal was decapitated. Brain tissue was centrifuged after flushing blood out of the brain. Determination of FITC-PACAP-TAT and FITC-PACAP was done via fluorimetry.	Uptake of TAT-PACAP38 was 6.55%inj and 2.77%inj for PACAP38.	TAT increased PACAP38s ability to cross the BBB 2.5-fold.

PTS-6 protein transport system-6, BBB blood-brain barrier, PACAP pituitary adenylate cyclase-activating polypeptide, BSCB blood-spinal cord barrier, MCA middle cerebral artery, ICR outbred mouse, SAMP8 a mouse model of Alzheimer, where altered BBB to other substances is known, LPS lipopolysaccharide (used to disrupt the BBB), TAT a peptide derived from the human immunodeficiency virus 1, MMA middle meningeal artery