Effect of controlled cortical impact on the passage of pituitary adenylate cyclase activating polypeptide (PACAP) across the blood-brain barrier

Abstract

Injuries to the central nervous system can affect the blood-brain barrier (BBB), including disruption and influencing peptide transport across the BBB. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neurotrophic and neuroprotective peptide currently being investigated for its therapeutic role following injury to the central nervous system and can cross the BBB in a saturable manner. The goal of the current study was to investigate for the first time PACAP38 uptake by the brain following traumatic brain injury (TBI). Using radioactively labeled PACAP38, we measured the levels of PACAP38 present in the injured, ipsilateral cortex in Sham-treated mice compared to mice receiving a controlled cortical impact (CCI), a model of TBI. Experiments were conducted at 6 different time points (from 2 hours up to 4 weeks) following CCI to determine temporal changes in PACAP38 transport. PACAP38 uptake was increased at 2 and 72 hours post-CCI compared to Sham. We did not detect changes in PACAP38 uptake in the contralateral cortex and cerebellum between Sham and CCI-treatment. The rate of PACAP38 transport into the ipsilateral cortex following CCI was increased 3.6-fold 72 hours after compared to 2 hours post-CCI. In addition, the rate of transport into the cerebellum was greater than that of the cortices. The data presented here shows PACAP38 transport is temporally altered following CCI-treatment and PACAP38 uptake is greater in the cerebellum compared to the cortices.

1. Introduction

Traumatic brain injury (TBI) is a devastating injury that can lead to long-lasting detrimental effects not only on mobility but also on cognitive capabilities. It is defined as brain damage due to an external mechanical force and is divided into a primary and secondary brain injury. The primary injury occurs at the time of impact and is impossible to prevent. The secondary brain injury occurs over time in response to a cascade of molecular changes occurring due to the initial insult. This secondary, neuroinflammatory injury can be limited if intervention occurs early. Controlled cortical impact (CCI) is a common TBI model used in rodents that delivers a focal injury that is highly reproducible in the amount of time, velocity, and depth of the impact [1]. In order to protect the brain from the detrimental effects of TBI, therapeutics must navigate the blood-brain barrier (BBB).

Pituitary adenylate cyclase-activating polypeptide (PACAP) is one such peptide that has been investigated for protection following various CNS injuries including TBI [2] and stroke [3]. It was first isolated as a neuropeptide [4] and belongs to the vasoactive intestinal polypeptide/secretin/glucagon family. There are two forms of PACAP varying in the amino acid length, PACAP27 and PACAP38. PACAP38 is more resistant to degradation in brain and efflux out of the brain compared to PACAP27. PACAP has neuroprotective capabilities and has been studied extensively in the context of ischemic stress [5]. Not only can it inhibit neuronal cell death [6] but it can also enhance endogenous antioxidant activity [7]. Following stroke, neurogenesis in the hippocampus was found to be dependent on PACAP38 [3]. Direct delivery of PACAP38 to the lateral cerebral ventricle prior to TBI in rats prevented motor deficits and prevented neural necrosis in the hippocampus [8]. Following TBI in humans, plasma and cerebrospinal fluid (CSF) PACAP38 levels are increased [9] suggesting PACAP38 transport across the BBB increases following TBI.

Under non-pathological conditions, PACAP38 transport occurs across the mouse BBB at a rate of 2.86 ± 0.57 μL/g-min [10]. The half-time disappearance of PACAP38 from the blood is very short at 0.82 min. Addition of more than 20 μg/mouse of unlabeled PACAP38 results in almost complete inhibition of PACAP38 entry into the brain, indicative of a saturable transport system. The transporter for PACAP38 is thought to be peptide transport system 6 (PTS-6) [10].

Changes in PACAP38 transport across the BBB occurs after central nervous system (CNS) injury whether via spinal cord injury [11] or middle cerebral artery occlusion (MCAO) [12] and were not confined to the site of injury either, suggesting widespread adaptations. Thirty minutes after spinal cord injury resulted in a decrease in PACAP38 BBB transport while seven days after injury resulted in increased transport at the site of injury in the lumbar spine [11], suggesting PACAP38 transport is temporally regulated. Beginning 6 hours following MCAO, PACAP38 transport across the BBB is decreased and transport returns to normal within 48 hours [12]. This group also showed treatment with PACAP38 8–12 hours after stroke had no neuroprotective effect [13]. Therefore, it is likely PACAP38 transport across the BBB is highly regulated, with alterations in the transport rate possibly based on the therapeutic requirements of the brain. In the current study, we sought to determine whether direct injury to the brain in a model of TBI alters the transport of PACAP38 into the brain.

2. Materials and Methods

2.1 Animal Use

Male CD-1 mice (8–10 weeks old) purchased from Charles River Laboratories (Seattle, WA) were used for all studies. Mice had ad libitum access to food and water and kept on a 12/12 hour light/dark cycle until study. All animal studies were approved by the Institutional Animal Care and Use Committee and performed at a facility that is approved by Association for Assessment and Accreditation of Laboratory Animal Care International.

2.2 Radioactive Labeling

The 38-amino acid form of PACAP (Bachem, Torrance, CA) was radioactively labeled with 0.5 mCi [¹²⁵I] (Perkin Elmer, Waltham, MA) by the lactoperoxidase (Sigma-Aldrich, St. Louis, MO) method. Briefly, 10 μg of PACAP38 was mixed with 30 μL of 0.4 M sodium acetate (pH5.6), 10 μL of lactoperoxidase (10 μg/mL) and 0.5 mCi [¹²⁵I]. The reaction was started by adding 200 ng of H₂O₂ in a volume of 10 μL and 10 min later, an additional 200 ng of H₂O₂ in a volume of 10 μL was added. At the end of the second 10-min period, the mixture was purified with high performance liquid chromatography on a C-18 column and 1 mL fractions collected. For the single time point transport experiments, 10 μg albumin (Sigma-Aldrich, St. Louis, MO) was radioactively labeled with 2 mCi [¹³¹I] Perkin Elmer, Waltham, WA) using the chloramine T (Sigma-Aldrich, St. Louis, MO) method. The reaction was started by adding 10 μg chloramine-T in 0.25 M chloride-free sodium phosphate buffer, pH 7.5. After 1 min, the reaction was terminated by adding 100 μg of sodium metabisulfite. Free I was separated from the labeled PACAP (I-PACAP38) or the labeled albumin (I-Alb) on a column of Sephadex G-10. For the multiple-time regression studies and capillary depletion, albumin was labeled with [^99mTc] (GE Healthcare, Seattle, WA). Briefly, 1 mg albumin was combined with 120 μg stannous tartrate and 20 μL 1M HCl in 500 mL deionized water. Addition of 1 mCi [^99mTc] began the 20 min reaction. Radioactively labeled albumin (Tc-Alb) was purified on a column of Sephadex G-10 (Sigma-Aldrich, St. Louis, MO). Protein labeling by iodine and technetium isotopes was characterized by 30% trichloroacetic acid precipitation. Greater than 90% radioactivity in the precipitated fraction was consistently observed for I-PACAP38, I-Alb, and Tc-Alb.

2.3 CCI Administration

Male CD-1 mice were placed under 2–4% isoflurane (0.5 L/min) to shave the head and maintained under anesthesia using a nose cone once placed in a stereotactic frame. Using sterile technique, the skull was exposed by a midline incision and skin retracted. A 4 mm diameter opening was made in the skull (3 mm lateral and 2 mm caudal of the bregma), exposing the right parietotemporal cortex. The bone was removed. The cortex was compressed by using the impactor device to deliver a 3 mm diameter flat tip weight at a velocity of 5.82 m/s to a depth of 1.2 mm for a duration of 47 ms at a driving pressure of 73 psi. The skin was placed back into position and cemented, the isoflurane stopped, and the mouse was placed on a heating pad until regaining consciousness, before being placed back in the home cage. Sham mice went through the procedure except for delivery of impact. Transport of I-PACAP38 and I-Alb was then measured 2 hours, 24 hours, 72 hours, 1 week, 2 weeks, or 4 weeks post-procedure (either Sham or CCI). Transport was also measured in control mice that were not subject to either Sham or CCI treatment on each study day to determine if there was an effect of Sham treatment or day-to-day variance (No-Sham Controls).

2.4 Measurement of I-PACAP38 and I-Alb Levels in Brain

Mice were anesthetized with an intraperitoneal injection of 0.15 mL of 40% urethane. The left jugular vein and right carotid artery were exposed. Mice were given an injection into the jugular vein of 0.2 mL of 0.1% BSA in lactated Ringer’s solution (BSA-LR) containing 1×10⁶ cpm I-PACAP38 and 5×10⁵ cpm I-Alb. The solution circulated for 5 min prior to blood collection and brain extraction. Brains were dissected into ipsilateral cortex, contralateral cortex, cerebellum, and remaining brain and weighed. The arterial blood was centrifuged at 5400g for 10 min at 4°C, and the serum was collected. The levels of radioactivity in serum (50 μL) and brain regions were counted in a gamma counter. Brain/serum ratios (μL/g) were calculated using the formula:

$$\text{Ratio}=(\text{cpm}/\mathrm{g}\text{of}\text{tissue})/(\text{cpm}/\mu \mathrm{L}\text{of}\text{serum})$$

Levels were corrected for the vascular space by subtracting the brain/serum ratio of I-Alb. Whole brain values are calculated by adding together the CPMs of each brain region and dividing by the sum total of the individual brain region weights.

Due to the large number of mice in this study, the experiments were spread out over multiple days. Shams and No-Sham Controls were performed with each experiment to account for any day-to-day differences. BBB disruption as determined with I-Alb and I-PACAP38 transport values are expressed as % Control (No-Sham Controls) for the respective day.

2.5 Measurement of Brain Influx

Multiple-time regression analysis [14, 15] was used to determine the blood-to-brain unidirectional influx rate (K_i) of I-PACAP38 at two time points post-CCI: 2 and 72 hours. Again, control mice receiving that did not have Sham or CCI treatment were used to determine effects due to Sham treatment and control for day-to-day variation. Mice were anesthetized with an intraperitoneal injection of 0.15 mL of 40% urethane. The left jugular vein and right carotid artery were exposed. Mice were given an injection into the jugular vein of 0.2 mL of BSA-LR containing 1×10⁶ cpm of I-PACAP38 and 5×10⁵ cpm of Tc-Alb. Blood from the carotid artery was collected between 1 to 10 min after intravenous injection, and then the mice were immediately decapitated and the whole brain was removed, dissected, and weighed. The arterial blood was centrifuged at 5400g for 10 min at 4°C, and the serum was collected. The levels of radioactivity in serum (50 μL) and brain were counted in a gamma counter. The brain/serum ratios (μL/g) of I-PACAP38 in each gram of brain region was calculated separately and were plotted against their respective exposure times (Expt). Exposure time was calculated from the following formula:

$$\text{Am}/\text{Cpt}=K_i[{\int }_0^t\text{Cp}(t)\mathrm{d}t]/\mathrm{C}\mathrm{p}(t)+V_i$$

where Am is cpm/g of brain, Cpt is cpm/μL of arterial serum at time t, and Expt (min) is measured by the term

$$\left[{\int }_0^t\text{Cp}(t)\mathrm{d}t\right]/\mathrm{C}\mathrm{p}(t)$$

Expt corrects for the clearance of I-PACAP38 from blood and gives the time that would have elapsed if clearance did not occur. The linear portion of the relation between the brain/serum ratios versus Expt was used to calculate K_i (μL/g-min) and initial volume of distribution for brain (V_i; μL/g). The slope of the linearity measures K_i and is reported with its error term. The y-intercept of the linearity measures V_i, the initial volume of distribution in brain at t = 0. To calculate the %Inj/g for each time point, the V_i was subtracted from the brain/serum ratio of I-PACAP38 and multiplied by the %Inj/μL.

2.6 Statistics

All statistical analyses were performed using Prism 6.0 (GraphPad Software Inc., San Diego, CA). Means are reported with their standard error terms and compared by one-way analysis of variance (ANOVA) followed by Newman-Keuls post-test. Two means were compared by t test analysis. Linear regression lines were compared statistically with the Prism 6.0 software package.

3. Results

3.1 Brain Weight Changes with CCI

Brain weight was measured in the brain regions collected: ipsilateral cortex, contralateral cortex, cerebellum, and the sum of these to reflect whole brain. There was a significant decrease in whole brain weight at 1, 2, and 4 weeks following CCI compared to Sham (Fig 1A). There was a decrease in the weight of the ipsilateral cortex at 1 and 2 weeks after CCI compared to Sham animals (Fig 1B). There were no significant decreases in weight of the contralateral cortex or cerebellum (data not shown).

Figure 1. Effect of CCI on brain weight.

CCI (black bars) resulted in a decrease in A) whole brain weight at 1, 2, and 4w compared to Sham (white bars) and in the B) ipsilateral cortex at 1 and 2w. Data represents n=6–13 and expressed as mean ± SEM. (*p<0.05 as marked, t test)

3.2 Measurement of Vascular Space

Levels of I-Alb were measured to properly correct the levels of I-PACAP38 present in the brain regions. I-Alb was not altered due to CCI at any time point compared to the Sham in the whole brain or cerebellum after 5 min of circulation of the radioactive material (data not shown). Although the ipsilateral cortex did not exhibit changes in I-Alb levels, the contralateral cortex had an increase in I-Alb levels in the Sham-treated group compared to CCI 24 hours post-procedure (Fig 2). Such increases usually reflect either BBB leakage, capillary recruitment, or vasodilation.

Figure 2. Measurement of Vascular Space.

There was no effect of CCI (black bars) on the permeability to I-Alb in the A) ipsilateral or B) contralateral cortex. However, there was a significant increase in the % Brain/Serum Ratio of I-Alb in the contralateral cortex of the Sham group at 24h compared to CCI (*p<0.05, t test). Data are expressed relative to the daily No-Sham controls as mean ± SEM. Data represents n=6–13.

3.3 I-PACAP38 Levels after CCI

Levels of I-PACAP38 present in the brain after various times post-CCI were measured and compared to Sham-treated animals. The results are expressed relative to the daily No-Sham Controls (dotted line) to control for day-to-day variability. In whole brain, there was a 36% increase in I-PACAP38 uptake 72 hours after CCI compared to Sham (Fig 3A). Within the ipsilateral cortex, there was a 53% increase in I-PACAP38 uptake 2 hours after CCI compared to Sham (Fig 3B). In this same region, Sham groups at 2 hours and 24 hours also had changes in I-PACAP38 transport compared to the No-Sham Controls (decreased and increased, respectively). There was no significant change in I-PACAP38 uptake in the contralateral cortex or cerebellum (data not shown).

Figure 3. I-PACAP38 Levels after CCI.

I-PACAP38 levels increased post-CCI (black bars) compared to Sham (white bars) in A) whole brain at 72h and in the B) ipsilateral cortex at 2h (* = p<0.05, t test). Also in the ipsilateral cortex, I-PACAP38 levels in the Sham groups compared to the No-Sham Controls (dashed line) decreased at 2h and increased at 24h (Ψ = p<0.05 vs No-Sham Controls, one-way ANOVA). Data are expressed relative to the daily No-Sham controls as mean ± SEM and have been adjusted for I-Alb. Data represents n=5–12.

3.4 I-PACAP38 Pharmacokinetics across the BBB

As we only detected differences between Sham and CCI in brain I-PACAP38 levels at 2 and 72 hours post-CCI, we chose to measure the transport rate of I-PACAP38 at these time points to determine if the altered level of I-PACAP38 was due to a change in the transport rate. We first needed to verify the I-PACAP38 measured in the brain was present in the parenchyma rather than being completely sequestered by the capillaries. I-PACAP38 was indeed able to cross the endothelial cells and enter the brain parenchyma (data not shown).

Using multiple-time linear regression analysis, we did not observe differences in the transport rate of I-PACAP38 between Sham and CCI at either 2 or 72 hours (Table I, K_i). The average rate of transport into the whole brain for all groups was 2.98 μL/g-min. However, the level of I-PACAP38 binding to the endothelium (V_i) 72 hours post-procedure was different in whole brain, ipsilateral cortex, and cerebellum between the treatment groups (Table I). In whole brain, CCI increased the V_i compared to Sham.

Table I. Pharmacokinetics of I-PACAP38 BBB Transport.

Transport rate (K_i) did not vary between Sham and CCI at either 2 or 72 hours due to treatment. However, the level of PACAP38 vascular binding (V_i) did significantly differ between the groups 72 hours post-procedure in the whole brain, ipsilateral cortex, and cerebellum.

	2 Hour				72 Hour
Whole Brain	Ki (μL/g-min)	r	p	Vi (μL/g)	Ki (μL/g-min)	r	p	Vi (μL/g)
No-Sham Controls	2.075 ± 0.41	0.860	<0.001	12.19 ± 3.21	2.620 ± 0.70	0.764	<0.01	11.16 ± 8.20*
Sham	3.008 ± 0.40	0.936	<0.0001	5.148 ± 4.47	3.867 ± 0.82	0.858	<0.01	18.90 ± 8.84*
CCI	2.177 ± 0.40	0.889	<0.001	8.662 ± 4.67	4.149 ± 1.05	0.797	<0.01	27.24 ± 12.87*
Ipsilateral Cortex	Ki (μL/g-min)	r	p	Vi (μL/g)	Ki (μL/g-min)	r	p	Vi (μL/g)
No-Sham Controls	2.344 ± 0.40	0.892	<0.001	9.999 ± 3.11	2.546 ± 0.86	0.701	<0.05	11.02 ± 10.17*
Sham	2.501 ± 0.67	0.795	<0.01	5.778 ± 7.52	3.337 ± 1.12	0.724	<0.05	16.57 ± 12.13*
CCI	1.338 ± 0.54	0.658	<0.05	9.497 ± 6.37	4.874 ± 1.07	0.835	<0.01	12.14 ± 13.18*
Contralateral Cortex	Ki (μL/g-min)	r	p	Vi (μL/g)	Ki (μL/g-min)	r	p	Vi (μL/g)
No-Sham Controls	2.063 ± 0.65	0.729	<0.05	8.050 ± 5.06	2.787 ± 0.52	0.874	<0.001	8.971 ± 6.08
Sham	2.642 ± 0.36	0.932	<0.0001	4.903 ± 4.05	2.706 ± 1.15	0.641	<0.05	14.69 ± 12.38
CCI	1.876 ± 0.56	0.763	<0.05	7.923 ± 6.62	4.222 ± 0.94	0.832	<0.01	7.614 ± 11.53
Cerebellum	Ki (μL/g-min)	r	p	Vi (μL/g)	Ki (μL/g-min)	r	p	Vi (μL/g)
No-Sham Controls	3.768 ± 0.92	0.859	<0.01	24.96 ± 5.59	3.698 ± 1.15	0.797	<0.05	20.84 ± 14.72*
Sham	5.682 ± 1.06	0.884	<0.001	5.654 ± 11.85	6.699 ± 2.37	0.731	<0.05	46.56 ± 26.90*
CCI	3.661 ± 0.94	0.811	<0.01	16.39 ± 11.02	7.701 ± 1.87	0.808	<0.01	19.31 ± 23.00*

^*p<0.05 versus other procedures

Although there were not differences in transport due to CCI treatment, there were temporal and regional differences in PACAP38 transport (Table I). There was a 2–3 fold increase in the rate of transport at 72 hours post-CCI compared to the 2 hour time point (Fig 4). In addition, since investigators are beginning to find changes within the cerebellum following TBI, we wanted to determine if there were regional differences in the level of I-PACAP38 in this brain region. Indeed, the cerebellum has a nearly 2-fold greater transport rate for I-PACAP38 compared to the cortices and whole brain (Table I).

Figure 4. I-PACAP38 transport rate between the 2 hour and 72 hour CCI groups.

The rate of PACAP38 transport across the BBB was increased 72 hours post-CCI in the ipsilateral and contralateral cortex compared to 2 hours post-CCI. (* = p<0.05). Data represents n=9–11/group.

For therapeutic purposes, calculating the percent of the injected dose taken up per gram of brain (%Inj/g of brain) is often useful. This calculation incorporates both transport rate and peripheral pharmacokinetic factors. The maximum percent of I-PACAP38 present in each gram of brain tissue in whole brain did not differ between the treatment groups 2 hours post-procedure (Table II). However, 72 hours post-CCI results in nearly a doubling of I-PACAP38 in the ipsilateral cortex compared to Sham. In addition, the cerebellum, by far, has the greatest %Inj/g (average of 0.37 %Inj/g) compared to the whole brain.

Table 2. Maximum %Injection/gram I-PACAP38.

The level of I-PACAP38 present per gram of tissue was corrected for vascular binding (V_i) and expressed as a percentage of the amount I-PACAP38 injected. The maximum value for each group is presented with the range of values for the entire time course of 1–10 min in parentheses. n=10–11/group

Maximum %Injection/gram
Whole Brain	2 Hours	72 Hours
Sham	0.181 (0.045–0.181)	0.178 (−0.021–0.178)
CCI	0.147 (0.053–0.147)	0.254 (0.005–0.254)
Ipsilateral Cortex	2 Hours	72 Hours
Sham	0.143 (0.015–0.143)	0.178 (−0.023–0.178)
CCI	0.142 (0.012–0.142)	0.352 (0.024–0.352)
Contralateral Cortex	2 Hours	72 Hours
Sham	0.159 (0.014–0.159)	0.159 (−0.033–0.159)
CCI	0.142 (0.019–0.142)	0.243 (0.020–0.243)
Cerebellum	2 Hours	72 Hours
Sham	0.311 (0.005–0.311)	0.379 (−0.125–0.379)
CCI	0.261 (0.025–0.261)	0.468 (0.005–0.468)

4. Discussion

The major findings of these studies suggest PACAP38 transport across the BBB is not dramatically altered due to CCI. This contrasts to changes in PACAP transport with spinal cord injury [11] and stroke [6], but is similar to that found after cardiac infarction [16]. There were slight changes due to procedure (No-Sham vs Sham vs CCI), time post-procedure (2 hours – 4 weeks), and region (cerebellum vs ipsi- and contralateral cortex). These results are surprising given the dramatic injury to the CNS the CCI model delivers. We investigated 6 different time points in case the changes in PACAP38 were transient after injury and 3 key brain regions implicated in TBI to make sure we did not miss regional differences in transport. However, the differences between the Sham and CCI groups were minimal.

Beginning one week post-CCI, we observed a decrease in the weight of the ipsilateral cortex. Whether or not this is due to apoptosis or cell shrinkage, we did not investigate. It has been shown white matter atrophy occurs 4 weeks post-CCI [17]. We cannot conclude if there was edema as the wet and dry weight of the brain regions were not measured. Interestingly, the contralateral cortex, which did not have a change in brain weight, exhibited an increased level of I-Alb 24 hours after Sham treatment. This response in the contralateral cortex is likely due to an indirect or sympathetic effect of Sham treatment on the ipsilateral cortex. The increased level of I-Alb compared to CCI allows us to accurately measure the level of I-PACAP38 present in the brain region.

The molecular effects of PACAP38 in the CNS following CCI injury have been previously investigated [7]. Miyamoto et al showed that PACAP38 administration immediately following CCI was able to prevent neurodegeneration and decrease levels of nitrotyrosine likely due to a concurrent increase in antioxidant capacity 24 hours following injury. Our data shows I-PACAP38 levels in the ipsilateral cortex are increased at 2 hours following CCI and 24 hours following Sham. In addition, whole brain I-PACAP38 levels were increased 72 hours following CCI. Interestingly, Sham-treated animals had a bidirectional change in I-PACAP38 level in the ipsilateral cortex at the two earliest time points (2 and 24 hours) post-procedure compared to No-Sham Controls. It is unclear why Sham treatment might result in changes in I-PACAP38 transport at these time points. It is possible to consider the Sham treatment group as a less severe form of TBI due to the surgery and manipulation of the skull and thus reveal effects of mild TBI on I-PACAP38 transport.

Our studies focused on the transport of peripheral radioactive PACAP38 across the BBB and into the brain. However, it is possible the beneficial effects of PACAP38 following TBI could be due to an increase in CNS expression of PACAP or a change in expression of the primary PACAP receptor, PAC1R. Indeed, PACAP mRNA is increased in the hippocampus and cortex at 72 hours following TBI in rats [18].

Previous studies have shown disruption of the BBB 72 hours following CCI [19, 20]. Therefore, we wanted to determine whether the changes in brain uptake of I-PACAP38 present at 2 and 72 hours post-CCI was due to alterations in transport kinetics. We measured the rate of transport at these time points in both Sham and CCI animals, comparing them to No-Sham Controls. The average rate of I-PACAP38 transport into whole brain in the No-Sham Controls was 2.35 μL/g-min, similar to the previously reported rate for mice of 2.86 μL/g-min [10]. There were no differences in the transport rate of I-PACAP38 between Sham and CCI at either time point. Therefore, the increased level of I-PACAP38 in CCI vs Sham presented in Fig 3 could be due to changes in binding of PACAP38 to the brain endothelial receptors or sequestration/degradation of PACAP38.

As there were no changes in rate of transport for the No-Sham Controls at either 2 or 72 hours, we investigated whether the time post-treatment impacted PACAP38 transport. The rate of transport was greater in the cortices 72 hours post-CCI compared to 2 hours. This data suggests I-PACAP38 transport is enhanced at this time point following TBI compared to the earlier 2 hour time point. This finding suggests potential temporal changes take place following TBI regarding PACAP38 transport.

In addition, we observed regional differences in the rate of I-PACAP38 transport across the BBB. The regional differences in I-PACAP38 transport rate have been investigated before [21]. Cerebellum transport rate is slightly higher than the cortex, similar to the results presented here. The cerebellum is significantly affected by TBI, specifically after blast injury, in regards to neuronal loss, lower glucose metabolism, and neuroinflammation [22].

In addition to the differences in the rate of I-PACAP38 transport across the BBB, the level of I-PACAP38 binding to the vascular endothelium was altered. This was most apparent change was in the whole brain levels 72 hours post-CCI. The Sham-treated group (18.90 μL/g) had a similar V_i to what has been previously reported for ICR mice (18.5 μL/g) [10]. However, the CCI group had a 1.4 fold increase in the V_i (27.24 μL/g) in the whole brain. It is unclear why this increase in V_i appears following CCI. One potential explanation could be molecular changes at the BBB in receptors for PACAP38 including vasoactive intestinal peptide receptor (VPAC) and PACAP type 1 receptor (PAC1) or changes in expression of PTS-6.

The actual amount of I-PACAP38 to enter the brain under non-pathological conditions has previously been calculated to be 0.119 %Inj/g in mice [23] and is similar to the value we calculated here of 0.15 %Inj/g. This compares favorably to the brain uptake of other therapeutics; for example, less than 0.02 %Inj/g of morphine enters the brain [24]. Although CCI does not appear to change this value 2 hours post-injury, levels in the ipsilateral cortex 72 hours post-CCI were nearly two times greater compared to Sham-treatment. In addition, the highest %Inj/g levels of I-PACAP38 were in the cerebellum, consistent with the increased rate of transport we observed in this brain region.

The data presented here suggests changes in I-PACAP38 transport following TBI are not as dramatic as following other CNS injuries such as stroke and spinal cord injury. There are temporal changes in I-PACAP38 transport at 72 hours compared to 2 hours post-CCI which could help guide therapeutic delivery. However, the molecular impact of therapeutic PACAP38 delivery 72 hours following CCI remain to be determined in addition to whether or not key PACAP38 receptors and transporters at the BBB are altered following TBI. The transport results obtained here will help guide future mechanistic investigations due to TBI on the transport of PACAP38.

HIGHLIGHTS.

• PACAP38 transport across the BBB is not dramatically altered following CCI.

• The ipsilateral cortex has increased PACAP38 levels 2h and 24h following CCI.

• There is no change in the rate of PACAP38 transport at these times.

• The cerebellum contains the greatest amount and fastest rate of PACAP38 transport.

Glossary

BBB

blood-brain barrier

CCI

controlled cortical impact

CNS

central nervous system

PACAP

pituitary adenylate cyclase-activating polypeptide

TBI

traumatic brain injury